Devices and methods for pharmacokinetic-based cell culture system

ABSTRACT

Devices, in vitro cell cultures, systems, and methods are provided for microscale cell culture analogous (CCA) device.

RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/286,493, filed Apr. 25, 2001, the entirety of which isincorporated herein by reference.

STATEMENT REGARDING GOVERNMENT RIGHTS

This invention was supported at least in part under grant numberNAG8-1372 from the National Aeronautics and Space Administration. TheU.S. Government may have certain rights in the invention.

FIELD OF THE INVENTION

The field of the invention is cell culture devices and methods of use.

BACKGROUND OF THE INVENTION

Pharmacokinetics is the study of the fate of pharmaceuticals and otherbiologically active compounds from the time they are introduced into thebody until they are eliminated. For example, the sequence of events foran oral drug can include absorption through the various mucosalsurfaces, distribution via the blood stream to various tissues,biotransformation in the liver and other tissues, action at the targetsite, and elimination of drug or metabolites in urine or bile.Pharmacokinetics provides a rational means of approaching the metabolismof a compound in a biological system. For reviews of pharmacokineticequations and models, see, for example, Poulin and Theil (2000) J PharmSci. 89(1):16-35; Slob et al. (1997) Crit Rev Toxicol. 27(3):261 -72;Haddad et al. (1996) Toxicol Lett. 85(2):113-26; Hoang (1995) ToxicolLett. 79(1-3):99-106; Knaak et al. (1995) Toxicol Lett. 79(1-3):87-98;and Ball and Schwartz (1994) Comput Biol Med. 24(4):269-76.

One of the fundamental challenges researchers face in drug,environmental, nutritional, consumer product safety, and toxicologystudies is the extrapolation of metabolic data and risk assessment fromin vitro cell culture assays to animals. Although some conclusions canbe drawn with the application of appropriate pharmacokinetic principles,there are still substantial limitations. One concern is that currentscreening assays utilize cells under conditions that do not replicatetheir function in their natural setting. The circulatory flow,interaction with other tissues, and other parameters associated with aphysiological response are not found in standard tissue culture formats.For example, in a macroscale cell culture analog (CCA) system, cells aregrown at the bottom of chambers. These systems have non-physiologicalhigh liquid-to-cell ratios, and have an unrealistic ratio of cell types(e.g., ratio of liver to lung cells). In a variant form of themacroscale CCA system the cells are grown on microcarrier beads. Thesesystems more closely resemble physiological conditions, but are stilldeficient because they do not mimic physiological conditions accuratelyenough for predictive studies. Therefore, the resulting assay data isnot based on the pattern of drug or toxin exposure that would be foundin an animal.

Within living beings, concentration, time and metabolism interact toinfluence the intensity and duration of a pharmacologic or toxicresponse. For example, in vivo the presence of liver function stronglyaffects drug metabolism and bioavailability. Elimination of an activedrug by the liver occurs by biotransformation and excretion.Biotransformation reactions include reactions catalyzed by thecytochrome P450 enzymes, which transform many chemically diverse drugs.A second biotransformation phase can add a hydrophilic group, such asglutathione, glucuronic acid or sulfate, to increase water solubilityand speed elimination through the kidneys.

While biotransformation can be beneficial, it may also have undesirableconsequences. Toxicity results from a complex interaction between acompound and the organism. During the process of biotransformation, theresulting metabolite can be more toxic than the parent compound. Thesingle-cell assays used by many for toxicity screening miss thesecomplex inter-cellular and inter-tissue effects.

Consequently, accurate prediction of human responsiveness to potentialpharmaceuticals is difficult, often unreliable, and invariablyexpensive. Traditional methods of predicting human response utilizesurrogates—typically either static, homogeneous in vitro cell cultureassays or in vivo animal studies. In vitro cell culture assays are oflimited value because they do not accurately mimic the complexenvironment a drug candidate is subjected to within a human and thuscannot accurately predict human risk. Similarly, while in vivo animaltesting can account for these complex inter-cellular and inter-tissueeffects not observable from in vitro cell-based assays, in vivo animalstudies are extremely expensive, labor-intensive, time consuming, andoften the results are of doubtful relevance when correlating human risk.

U.S. Pat. No. 5,612,188 issued to Shuler et al. describes amulticompartmental cell culture system. This culture system uses largecomponents, such as culture chambers, sensors, and pumps, which requirethe use of large quantities of culture media, cells and test compounds.This system is very expensive to operate and requires a large amount ofspace in which to operate. Because this system is on such a large scale,the physiological parameters vary considerably from those found in an invivo situation. It is impossible to accurately generate physiologicallyrealistic conditions at such a large scale.

The development of microscale screening assays and devices that canprovide better, faster and more efficient prediction of in vivo toxicityand clinical drug performance is of great interest in a number offields, and is addressed in the present invention. Such a microscaledevice would accurately produce physiologically realistic parameters andwould more closely model the desired in vivo system being tested.

SUMMARY OF THE INVENTION

Devices, in vitro cell cultures, and methods are provided for amicroscale cell culture analog (CCA) device. The devices of theinvention permit cells to be maintained in vitro, under conditions withpharmacokinetic parameter values similar to those found in vivo.Pharmacokinetic parameters of interest include interactions betweencells, liquid residence time, liquid to cell ratios, relative size oforgans, metabolism by cells, shear stress, and the like. By providing apharmacokinetic-based culture system that mimics the natural state ofcells, the predictive value and in vivo relevance of screening andtoxicity assays is enhanced.

The microscale culture device comprises a fluidic network of channelssegregated into discrete but interconnected chambers. The specificchamber geometry is designed to provide cellular interactions, liquidflow, and liquid residence parameters that correlate with those foundfor the corresponding cells, tissues, or organs in vivo. The fluidicsare designed to accurately represent primary elements of the circulatoryor lymphatic systems. In one embodiment, these components are integratedinto a chip format. The design and validation of these geometries isbased on a physiological-based pharmacokinetic (PBPK) model; amathematical model that represents the body as interconnectedcompartments representing different tissues.

The device can be seeded with the appropriate cells for each culturechamber. For example, a chamber designed to provide liverpharmacokinetic parameters is seeded with hepatocytes, and may be influid connection with adipocytes seeded in a chamber designed to providefat tissue pharmacokinetics. The result is a pharmacokinetic-based cellculture system that accurately represents, for example, the tissue sizeratio, tissue to blood volume ratio, drug residence time of the animalit is modeling.

In one embodiment, a system includes a first microscale culture deviceand a control instrument. The first microscale culture device has anumber of microscale chambers with geometries that simulate a pluralityof in vivo interactions with a culture medium, wherein each chamberincludes an inlet and an outlet for flow of the culture medium, and amicrofluidic channel interconnecting the chambers. Thecontrol-instrument is coupled to the first microscale culture device,and includes a computer to acquire data from, and controlpharmacokinetic parameters of, the first microscale culture device.

In another embodiment, a computer includes a microprocessor, a generalmemory, a non-volatile storage element, an input/output interface thatincludes an interface to a microscale culture device having one or moresensors, and computer software. The computer software is executable onthe microprocessor to analyze data from the sensors to measurephysiological events in a number of chambers of the microscale culturedevice, regulate fluid flow rates of a culture medium in the chambers ofthe microscale culture device, detect biological or toxicologicalreactions in the chambers of the microscale culture device, and upondetection, change one or more pharmacokinetic parameters of themicroscale culture device.

As used herein the singular forms “a” and “the” include plural referentsunless the context clearly dictates otherwise. For example, “a compound”refers to one or more of such compounds, while “the cell” includes aparticular cell as well as other family members and equivalents thereofas known to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of a system in accordance with the presentinvention.

FIG. 2 is a simplified perspective view of one embodiment of theexterior of the system of the present invention.

FIG. 3 is a detailed schematic view of another embodiment of the systemof the present invention.

FIG. 4 is a schematic view of yet another embodiment of the system ofthe present invention.

FIGS. 5A through 5G show steps used to fabricate a chip from plastic.FIG. 5A shows coating a silicon wafer with a positive photoresistmaterial. FIG. 5B shows exposing resist-coated silicon wafer to UV lightthrough a photomaterial. FIG. 5C shows developing the photoresistmaterial. FIG. 5D shows etching silicon. FIG. 5E shows striping thephotoresist material and evaporating gold, FIG. 5F shows electroplatingnickel. FIG. 5G shows removing silicon and embossing polymer.

FIG. 6 is a schematic view of still another embodiment of the system ofthe present invention.

FIG. 7 is a schematic detailing a computer associated with the chips.

FIG. 8 is a schematic showing more than one chip located within ahousing.

FIG. 9 is a schematic of a system that includes sets of chips fromdifferent housings.

FIG. 10 is a schematic of yet another embodiment of a chip.

FIG. 11 is an isometric partially exploded view of a system.

FIG. 12 is an isometric view of the steps for fabricating the chipassociated with the system shown in FIG. 11.

FIG. 13 is an isometric view of a single trough elastomeric portion of apump associated with the system shown in FIG. 11.

FIG. 14 is an isometric view of a multiple trough elastomeric portion ofa pump.

FIG. 15 is a schematic diagram of the four compartment chip.

FIG. 16 Tegafur dose response. Chips were seeded with HepG2-C3A cells inthe liver compartment and HCT-116 colon cancer cells in the targettissues compartment. The chips were treated with indicatedconcentrations of tegafur for 24 hours. The first graph (FIG. 16A) is aplot of percentage dead cells vs. tegafur or 5-FU concentration after 24hours of recirculation on the chip. The second graph (FIG. 16B) is asimilar dose response using a traditional in vitro cell culture assaywith HCT 116 cells using a 48 hour exposure. HCT-116 cells were seededon poly-lysine treated glass coverslips and exposed to either tegafur or5-FU at the indicated concentrations. After a 48 hr incubation,coverslips were treated as described above and the percentage of celldeath was determined.

FIG. 17A depicts a “first generation” three compartment device. FIG. 17Bshows a cross-sectional view of the device.

FIG. 18A depicts a “second generation” device. FIG. 18B depicts 5 μmtall ridges in a chamber, and FIG. 1C depicts 20 μm tall pillars in achamber.

FIG. 19 depicts a “third generation” device.

FIG. 20 is a flow diagram for a five compartment PBPK model CCA.

FIG. 21 depicts a human biochip prototype that contains compartments forlung, target tissues, and other tissues. The dimensions of thecompartments and channels are as follows:

Inlet: 1 mm by 1 mm

Liver: 3.2 mm wide by 4 mm long

Target Tissues: 2 mm wide by 2 mm long

Other Tissues: 340 μμm wide by 110 mm long

Outlet: 1 mm by 1 mm

Channel Connecting Liver to Y connection: 440 μμm wide Channel from Yconnection to Target Tissue: 100 μμm wide

FIG. 22 depicts a schematic drawing of the microscale chip system.

FIG. 23 depicts basal CYP expression levels for Hep G2, HepG2/C3A, andhuman liver. Std. error from 3 separate determinations.

FIG. 24A depicts HepG2/C3A growth curves in EMEM, DMEM, McCoy's andRPMI. FIG. 24B depicts HCT116 growth curves in EMEM, DMEM, McCoy's andRPMI. Standard error from 3 separate determinations.

FIG. 25 depicts RT-PCR determination of CYP isoforms expression inHepG2/C3A under different growth media conditions.

FIG. 26 depicts RT-PCR determination of CYP isoforms expression inHepG2/C3A grown on different substrates.

FIG. 27 depicts a human bio-chip prototype.

FIG. 28A is a block-diagram view illustrating a system for controlling amicroscale culture device, according to one embodiment of the presentinvention. FIG. 28B is a block-diagram view illustrating a system forcontrolling a microscale culture device, according to another embodimentof the present invention.

FIG. 29 is a flow-diagram view illustrating a computerized method fordynamically controlling a microscale culture device, according to oneembodiment of the present invention.

FIG. 30 is a block-diagram view illustrating a computer for controllinga microscale culture device, according to one embodiment of the presentinvention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present inventors have developed a microscale cell culture analog(CCA) system. Such a microscale CCA system has many advantages over theearlier macroscale systems. The microscale systems use smallerquantities of reagents, fewer cells (which allow the use of authenticprimary cells rather than cultured cells), are more physiologicallyrealistic (e.g., residence times, organ ratios, shear stresses), have alower device cost, and are smaller in size (multiple tests andstatistical analysis available). Moreover, multiple biosensors can beincorporated on the same chip.

In simplest terms, the chip of the present invention provides anaccurate in vitro surrogate of an whole animal or human. To accomplishthis, an initial design was produced using a physiological-basedpharmacokinetic (PBPK) model—a mathematical model that represents thebody as interconnected compartments specific for a particular organ.From the PBPK model and published empirical data, a lengthy andextensive development program resulted in a microscale device thataccurately mimics the known tissue size ratio, tissue to blood volumeratio, drug residence time, and other important physiological parametersof a whole animal or human. In essence, the chip technology of thepresent invention is a microscale model of a whole animal or human(˜1/100,000^(th) for human).

In operation, the device replicates a re-circulating multi-organ systemby segregating living cells into discrete, interconnected “organ”compartments (see e.g., FIG. 15). The fluidics are designed such thatthe primary elements of the circulatory system and the interactions ofthe organ systems are accurately mimicked. Each organ compartmentcontains a particular cell type carefully selected or engineered tomimic the primary function(s) of the corresponding whole organ (e.g.xenobiotic metabolism by the liver). The cell type may be adherent ornon-adherent and derived from standard cell culture lines or primarytissue. Human cells are used for human surrogates or cells from otherspecies as appropriate.

The organ compartments are connected by a re-circulating culture mediumthat acts as a “blood surrogate.” Test agents in the medium aredistributed and interact with the cells in the organ compartments muchas they would in the human body or whole animal. The effects of thesecompounds and/or their metabolites on the various cell types aredetected by measuring or monitoring key physiological events such ascell death, cell proliferation, differentiation, immune response, orperturbations in metabolism or signal transduction pathways. Inaddition, pharmacokinetic data can be determined by collecting andanalyzing aliquots of the culture medium for drug metabolites.

The microscale chip device of the present invention offers both the costand throughput advantages of traditional cell culture assays and alsothe high informational content of whole animal models. Unlike wholeanimal tests however, the chip is inexpensive and largely disposable.The low fluid volume (˜5 μl) of the device provides the high sensitivityand throughput characteristic of microfluidic devices. Moreover, thereadout of the device is highly flexible and assay independent—almostany cell type or assay can be used without modification. Numerousbiological assays based on optical interrogation and readout (e.g.,fluorescence, luminescence) are available, thus making real-timemonitoring feasible. Alternatively, standard pathology, biochemical,genomic or proteomic assays can be utilized directly as the system canbe designed to be fully compatible with the traditional coverslip (22mm×22 mm) or 96 well format. Further, genetically engineered cells canbe used for specialized end-user applications. In addition, “3D” chipscan be used to encompass additional compartments and modules to analyzegastrointestinal tract or blood-brain barrier absorption.

Unlike traditional in vitro assays, the chip of the present inventionmore closely mimics the complex multi-tissue (liver, lung, adipose,circulatory system, etc.) biology of the whole organism. Drug candidatesare exposed to a more realistic animal or human physiologicalenvironment thus providing higher and more accurate informationalcontent (e.g., absorption, distribution, bioaccumulation, metabolism,excretion, efficacy and toxicity) than typical in vitro assays. Thesebenefits directly affect the safety and efficacy predictions of drugleads and particularly, their prioritization before entering intoexpensive and time-consuming non-clinical or clinical trials. Thisprioritization increases drug development throughput, reduces the numberof animals needed for toxicological screening, decreases the costs ofnon-clinical studies, and increases the efficiency of clinical trials byallowing rapid and direct assessment of potential toxicity or lack ofefficacy prior to entering these trials.

These demonstrate some of the advantages of the chip technology of thepresent invention. In summary, acquisition of data is rapid whencompared to traditional in vitro cell culture assays, animal studies, orclinical trials. The data is also robust, providing highly predictivecontent not available from traditional in vitro assays. The chipplatform is designed such that it is fully compatible with existingassays—either in the standard coverslip or 96 well format. The deviceitself is configurable for any animal species or combination of multipleorgan compartments. Individual chips are priced cost-effectively asdisposables. Moreover, the low volume of the device further reducesreagent costs in screening potential compounds.

Unlike currently available technologies, the present chip system greatlyincreases the success rates not only at the clinical phase, but also inreducing the number of compounds that need to undergo pre-clinicaltesting. Consequently, a pharmaceutical company can (1) determine whichdrug candidates have the potential to be toxic to humans early in thedevelopment process; (2) better select the animal species that bestpredict human response; and (3) determine which drug candidate has thepotential to be efficacious. Thus, the chip of the present inventiongreatly increases the success rates and decrease the development time ofmarketable drugs.

Pharmokinetic-Based Microscale Culture Device

Devices, in vitro cell cultures, and methods are provided for a CCAdevice. The subject methods and devices provide a means whereby cellsare maintained in vitro in a physiologically representative environment,thereby improving the predictive value and in vivo relevance ofscreening and toxicity assays. A microscale pharmacokinetic culturedevice of the present invention is seeded with the appropriate cells foreach culture chamber, which culture system can then be used for compoundscreening, toxicity assays, models for development of cells of interest,models of infection kinetics, and the like. An -input variable, whichmay be, for example, a compound, sample, genetic sequence, pathogen,cell (such as a stem or progenitor cell), is added to an establishedculture system. Various cellular outputs may be assessed to determinethe response of the cells to the input variable, including pH of themedium, concentration of O₂ and CO₂ in the medium, expression ofproteins and other cellular markers, cell viability, or release ofcellular products into the culture medium.

The design and geometry of the culture substrate, or device, providesfor the unique growth conditions of the invention. Each device comprisesone or more chambers, which are interconnected by fluidic channels. Eachchamber may have a geometric configuration distinct from otherchamber(s) present on the device. For example, one embodiment of thedevice consists of chambers representing lung, liver, and other tissues(FIG. 18A). The lung chamber in this embodiment contains 5 μm tallridges in order to achieve realistic cell to liquid volume ratio andliquid residence time (FIG. 18B). The liver chamber in this embodimentcontains 20 μm tall pillars to achieve realistic cell to liquid volumeratio and liquid residence time (FIG. 18C). The device also comprisesinlet and outlet ports so that the culture medium can be circulated.

In one embodiment, the culture device is in a chip format, i.e., thechambers and fluidic channels are fabricated or molded from a fabricatedmaster, such that the device is formed either as a single unit or as amodular system with one or more chambers on separate units. Generallythe chip format is provided in a small scale, usually not more thanabout 10 cm on a side, or even not more than about 5 cm on a side. Itmay even be only about 2 cm on a side or smaller. In another example,the chip may be housed in a 96 well format in which the individual chipsare less than 0.9 cm×0.9 cm. The chambers and fluidic channels arecorrespondingly micro-scale in size.

In another embodiment, the culture device is in the form of anintegrated device consisting of a table-top instrument housing multiplemicroscale chips fabricated as disposable plastic polymer-basedcomponents. The instrument may consist of a base with depressions toaccommodate individual cell chips or alternatively, a single “chip” in astandard 96 well format (i.e., 96 individual chips in a 8×12 format).The instrument top, when closed seals the chips and provide fluidinterconnects. The instrument contains low volume pumps to re-circulatefluid to the chips and small 3-way valves with injection loops toprovide introduction of test compounds, or alternatively draws compoundsdirectly from a 96- or 384-well plate. Multiple compounds can beevaluated simultaneously for efficacy, toxicity, and/or metaboliteproduction using this instrument. The instrument may also integrateon-chip fluorescence detection for real-time physiology monitoring usingwell-characterized biomarkers.

The device may include a mechanism for obtaining signals from the cellsand culture medium. The signals from different chambers and channels canbe monitored in real time. For example, biosensors can be integrated orexternal to the device, which permit real-time readout of thephysiological status of the cells in the system.

The present invention provides an ideal system for high-throughputscreening to identify positive or negative response to a range ofsubstances such as, for example, pharmaceutical compositions, vaccinepreparations, cytotoxic chemicals, mutagens, cytokines, chemokines,growth factors, hormones, inhibitory compounds, chemotherapeutic agents,and a host of other compounds or factors. The substance to be tested canbe either naturally-occurring or synthetic, and can be organic orinorganic.

For example, the activity of a cytotoxic compound can be measured by itsability to damage or kill cells in culture. This may readily be assessedby vital staining techniques. The effect of growth/regulatory factorsmay be assessed by analyzing the cellular content of the matrix, e.g.,by total cell counts, and differential cell counts. This may beaccomplished using standard cytological and/or histological techniquesincluding the use of immunocytochemical techniques employing antibodiesthat define type-specific cellular antigens. The effect of various drugson normal cells cultured in the device may be assessed. For example,drugs that increase red blood cell formation can be tested on bonemarrow cultures. Drugs that affect cholesterol metabolism, e.g., bylowering cholesterol production, can be tested on a liver system.Cultures of tumor cells may be used as model systems to test, forexample, the efficacy of anti-tumor agents.

The device of the invention may be used as model systems for the studyof physiologic or pathologic conditions. For example, in a specificembodiment of the invention, a device can be used as a model for theblood-brain barrier, such a model system can be used to study thepenetration of substances through the blood-brain barrier. In anadditional embodiment, and not by way of limitation, a device containingmucosal epithelium may be used as a model system to study herpesvirus orpapillomavirus infection; such a model system can be used to test theefficacy of anti-viral medications.

The device of the present invention may also be used to aid in thediagnosis and treatment of malignancies and diseases. For example,biopsies of any tissue (e.g., bone marrow, skin, liver) may be takenfrom a patient suspected of having a malignancy. The patient's culturecan be used in vitro to screen cytotoxic and/or pharmaceutical compoundsin order to identify those that are most efficacious; i.e., those thatkill the malignant or diseased cells, yet spare the normal cells. Theseagents can then be used to therapeutically treat the patient.

In yet another embodiment of the invention, the device can be used invitro to produce biological products in high yield. For example, a cellthat naturally produces large quantities of a particular biologicalproduct (e.g., a growth factor, regulatory factor, peptide hormone,antibody), or a host cell genetically engineered to produce a foreigngene product, can be clonally expanded using the in vitro device. If atransformed cell excretes the gene product into the nutrient medium, theproduct may be readily isolated from the spent or conditioned mediumusing standard separation techniques (e.g., HPLC, column chromatography,electrophoretic techniques, to name but a few). A “bioreactor” can bedevised that would take advantage of the continuous flow method forfeeding cultures in vitro. Essentially, as fresh media is passed throughthe cultures in the device, the gene product will be washed out of theculture along with the cells released from the culture. The gene productcan be isolated (e.g., by HPLC column chromatography, electrophoresis)from the outflow of spent or conditioned media.

The present invention also provides a system for screening or measuringthe effects of various environmental conditions or compounds on abiological system. For example air or water conditions could be mimickedor varied in the device. The impact of different known or suspectedtoxic substances could be tested. The present invention further providesa system for screening consumer products, such as cosmetics, cleansers,or lotions. It also provides a system for determining the safety and/orefficacy of nutriceuticals, nutritional supplements, or food additives.The present invention could also be used as a miniature bioreactor orcellular production platform to produce cellular products in quantity.

Typical efficacy or toxicity experiments using the chip formatmicroscale culture device of the present invention are completed within24 to 48 hours or less depending on experimental design. Extendedexperiments, however, can be performed in order to test for the effectsof chronic exposure (e.g., genotoxicity, carcinogenicity, or latentdiseases).

The present invention provides novel devices, systems and methods as setforth within this specification. In general, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs, unless clearly indicated otherwise. For clarification, listedbelow are definitions for certain terms used herein to describe thepresent invention. These definitions apply to the terms as they are usedthroughout this specification, unless otherwise clearly indicated.

Definition of Terms

Pharmacokinetic-based culture system: An in vitro cell culture system,wherein the cells are maintained under conditions providingpharmacokinetic parameter values that model those found in vivo. Apharmacokinetic culture device comprises a fluidic network of channelssegregated into discrete but interconnected chambers, where the specificchamber geometry is designed to provide cellular interactions, liquidflow, and liquid residence parameters that correlate with those foundfor the corresponding cells, tissue, or organ system in vivo. The deviceis seeded with cells that are appropriate for conditions being modeled,e.g., liver cells in a liver-based culture chamber, lung cells in alung-based culture chamber, and the like, to provide the culture system.

The culture systems of the invention provide for at least onepharmacokinetic parameter value that is comparable to values obtainedfor the cell, tissue, or organ system of interest in vivo, preferably atleast two parameter values, and may provide for three or more comparableparameter values. Pharmacokinetic parameters of interest include, forexample, interactions between cells, liquid residence time, liquid tocell ratios, metabolism by cells, or shear stress.

By comparable values, it is meant that the actual values do not deviatemore than 25% from the theoretical values. For example, the calculatedor theoretical value for the liquid residence time in the lungcompartment for a rat is 2 seconds and the actual value measured in thelung cell culture chamber of a rat CCA device was 2.5±0.7 seconds.

The pharmacokinetic parameter value is obtained by using the equationsof a PBPK model. Such equations have been described in the art, forexample see Poulin and Theil (2000) J Pharm Sci. 89(1):16-35; Slob etal. (1997) Crit Rev Toxicol. 27(3):261-72; Haddad et al. (1996) ToxicolLett. 85(2):113-26; Hoang (1995) Toxicol Lett. 79(1-3):99-106; Knaak etal. (1995) Toxicol Lett. 79(1-3):87-98; and Ball and Schwartz (1994)Comput Biol Med. 24(4):269-76, herein incorporated by reference.Pharmacokinetic parameters can also be obtained from the publishedliterature, for example see Buckpitt et al., (1984) J. Pharmacol. Exp.Ther. 231:291-300; DelRaso (1993) Toxicol. Lett. 68:91-99; Haies et al.,(1981) Am. Rev. Respir. Dis. 123:533-541.

Specific physiologic parameters of interest include tissue or organliquid residence time, tissue or organ mass, liquid-to-cell volumeratio, cell shear stress, etc. Physiologically relevant parameter valuescan be obtained empirically according to conventional methods, or can beobtained from values known in the art and publicly available.Pharmacokinetic parameter values of interest are obtained for an animal,usually a mammal, although other animal models can also find use, e.g.,insects, fish, reptiles, or avians. Mammals include laboratory animals,e.g., mouse, rat, rabbit, or guinea pig mammals of economic value, e.g.,equine, ovine, caprine, bovine, canine, or feline; primates, includingmonkeys, apes, or humans; and the like. Different values may be obtainedand used for animals of different ages, e.g., fetal, neonatal, infant,child, adult, or elderly; and for different physiological states, e.g.,diseased, after contact with a pharmaceutically active agent, afterinfection, or under conditions of altered atmospheric pressure.

Information relevant to the pharmacokinetic parameter values, as well asmass balance equations applicable to various substances to be modeled inthe system, is optionally provided in a data processing component of theculture system, e.g., look-up tables in general purpose memory set asidefor storage, and the like. These equations representphysiologically-based pharmacokinetic models for variousbiological/chemical substances in systems.

Pharmacokinetic culture device: The culture device of the inventionprovides a substrate for cell growth. Each device comprises at least onechamber, usually at least two chambers, and may comprise three or morechambers, where the chambers are interconnected by fluidic channels. Thechambers can be on a single substrate or on different substrates.Preferably each chamber has a geometric configuration distinct fromother chamber(s) present on the device. The device contains a cover toseal the chambers and channels and comprises at least one inlet and oneoutlet port that allow for recirculation of the culture medium Thedevice contains a mechanism to pump the culture medium through thesystem. The culture medium is designed to maintain viability of thecultured cells. The device contains a mechanism by which test compoundscan be introduced to the system.

In one embodiment of the invention, the device is fabricated on amicroscale as a single unit of not more than about 2.5 cm in a side,preferably comprising at least two interconnected chambers. The twoorgan compartments are connected by a channel of from about 50-150 μmwide and 15-25 μm deep. For example, one chamber may represent the lung,comprising an interconnected array of parallel channels, usually atleast about 10 channels, preferably at least about 20 channels. Suchchannel may have typical microfluidic dimensions, e.g., about 30-50 μmwide, 5-15 μm deep and 3-7 mm long. Another compartment may representthe liver, comprising two or more parallel channels, usually from about50-150 μm wide, 15-25 μm deep and 5-15 cm long in a serpentine shape.

The device will usually include a mechanism for obtaining signals fromthe cells and culture medium. The signals from different chambers andchannels can be monitored in real time. For example, biosensors can beintegrated or external to the device, which permit real-time readout ofthe physiological status of the cells in the system.

The pharmacokinetic culture device of the present invention may beprovided as a chip or substrate. In addition to enhancing the fluiddynamics, such microsystems save on space, particularly when used inhighly parallel systems, and can be produced inexpensively. The culturedevice can be formed from a polymer such as but not limited topolystyrene, and disposed of after one use, eliminating the need forsterilization. As a result, the in vitro subsystem can be producedinexpensively and widely used. In addition, the cells may be grown in athree-dimensional manner, e.g., to form a tube, which more closelyreplicates the in vivo environment.

To model the metabolic response of an animal for any particular agent, abank of parallel or multiplex arrays comprising a plurality (i.e., atleast two) of the cell culture systems, where each system can beidentical, or can be varied with predetermined parameter values or inputagents and concentrations. The array may comprise at least about 10, ormay even be as many as 100 or more systems. Advantageously, the cellculture systems on microchips can be housed within a single chamber sothat all the cell culture systems under are exposed to the sameconditions during an assay.

Alternatively, multiple chips may be interconnected to form a singledevice, e.g., to mimic gastrointestinal barriers or the blood brainbarrier.

Cells: Cells for use in the assays of the invention can be an organism,a single cell type derived from an organism, and can be a mixture ofcell types, as is typical of in vivo situations. The culture conditionsmay include, for example, temperature, pH, presence of factors, presenceof other cell types, and the like. A variety of animal cells can beused, including any of the animals for which pharmacokinetic parametervalues can be obtained, as previously described.

The invention is suitable for use with any cell type, including primarycells, stem cells, progenitor cells, normal, genetically-modified,genetically altered, immortalized, and transformed cell lines. Thepresent invention is suitable for use with single cell types or celllines, or with combinations of different cell types. Preferably thecultured cells maintain the ability to respond to stimuli that elicit aresponse in their naturally occurring counterparts. These may be derivedfrom all sources such as eukaryotic or prokaryotic cells. The eukaryoticcells can be plant, or animal in nature, such as human, simian, orrodent. They may be of any tissue type (e.g., heart, stomach, kidney,intestine, lung, liver, fat, bone, cartilage, skeletal muscle, smoothmuscle, cardiac muscle, bone marrow, muscle, brain, pancreas), and celltype (e.g., epithelial, endothelial, mesenchymal, adipocyte,hematopoietic). Further, a cross-section of tissue or an organ can beused. For example, a cross-section of an artery, vein, gastrointestinaltract, esophagus, or colon could be used.

In addition, cells that have been genetically altered or modified so asto contain a non-native “recombinant” (also called “exogenous”) nucleicacid sequence, or modified by antisense technology to provide a gain orloss of genetic function may be utilized with the invention. Methods forgenerating genetically modified cells are known in the art, see forexample “Current Protocols in Molecular Biology,” Ausubel et al., eds,John Wiley & Sons, New York, N.Y., 2000. The cells could be terminallydifferentiated or undifferentiated, such as a stem cell. The cells ofthe present invention could be cultured cells from a variety ofgenetically diverse individuals who may respond differently to biologicand pharmacologic agents. Genetic diversity can have indirect and directeffects on disease susceptibility. In a direct case, even a singlenucleotide change, resulting in a single nucleotide polymorphism (SNP),can alter the amino acid sequence of a protein and directly contributeto disease or disease susceptibility. For example, certainAPO-lipoprotein E genotypes have been associated with onset andprogression of Alzheimer's disease in some individuals.

When certain polymorphisms are associated with a particular diseasephenotype, cells from individuals identified as carriers of thepolymorphism can be studied for developmental anomalies, using cellsfrom non-carriers as a control. The present invention provide anexperimental system for studying developmental anomalies associated withparticular genetic disease presentations since several different celltypes can be studied simultaneously, and linked to related cells. Forexample, neuronal precursors, glial cells, or other cells of neuralorigin, can be used in a device to characterize the cellular effects ofa compound on the nervous system. Also, systems can be set up so thatcells can be studied to identify genetic elements that affect drugsensitivity, chemokine and cytokine response, response to growthfactors, hormones, and inhibitors, as well as responses to changes inreceptor expression and/or function. This information can be invaluablein designing treatment methodologies for diseases of genetic origin orfor which there is a genetic predisposition.

In one embodiment of the invention, the cells are involved in thedetoxification and metabolism of pharmaceutically active compounds,e.g., liver cells, including hepatocytes; kidney cells including tubulecells; fat cells including adipocytes that can retain organic compoundsfor long periods of time. These cells may be combined in a culturesystem with cells such as lung cells, which are involved in respirationand oxygenation processes. These cells may also be combined with cellsthat are particularly sensitive to damage from an agent of interest,e.g., gut epithelial cells, bone marrow cells, and other normallyrapidly dividing cells for agents that affect cell division. Neuralcells may be present to monitor for the effect of an agent forneurotoxicity, and the like.

The growth characteristics of tumors, and the response of surroundingtissues and the immune system to tumor growth are also of interest.Degenerative diseases, including affected tissues and surrounding areasmay be exploited to determine both the response of the affected tissue,and the interactions with other parts of the body.

The term “environment” or “culture condition” encompasses cells, media,factors, time and temperature. Environments may also include drugs andother compounds, particular atmospheric conditions, pH, saltcomposition, minerals, etc. Cell culturing is typically performed in asterile environment mimicking physiological conditions, for example, at37° C. in an incubator containing a humidified 92-95% air/5-8% CO₂atmosphere. Cell culturing may be carried out in nutrient mixturescontaining undefined biological fluids such a fetal calf serum, or mediathat is fully defined and serum free. A variety of culture media areknown in the art and are commercially available.

The term “physiological conditions” as used herein is defined to meanthat the cell culturing conditions are very specifically monitored tomimic as closely as possible the natural tissue conditions for aparticular type of cell in vivo. These conditions include suchparameters as liquid residence time (i.e., the time that a liquid staysin an organ); cell to blood volume ratio, sheer stress on the cells,size of compartment comparable to natural organ.

Screening Assays: Drugs, toxins, cells, pathogens, samples, etc., hereinreferred to generically as “input variables” are screened for biologicalactivity by adding to the pharmacokinetic-based culture system, and thenassessing the cultured cells for changes in output variables ofinterest, e.g., consumption of O₂, production of CO₂, cell viability, orexpression of proteins of interest. The input variables are typicallyadded in solution, or readily soluble form, to the medium of cells inculture. The input variables may be added using a flow through system,or alternatively, adding a bolus to an otherwise static solution. In aflow-through system, two fluids are used, where one is a physiologicallyneutral solution, and the other is the same solution with the testcompound added. The first fluid is passed over the cells, followed bythe second. In a single solution method, a bolus of the test inputvariables is added to the volume of medium surrounding the cells. Theoverall composition of the culture medium should not changesignificantly with the addition of the bolus, or between the twosolutions in a flow through method.

Preferred input variables formulations do not include additionalcomponents, such as preservatives, that have a significant effect on theoverall formulation. Thus, preferred formulations include a biologicallyactive agent and a physiologically acceptable carrier, e.g., water,ethanol, or DMSO. However, if an agent is liquid without an excipient,the formulation may be only the compound itself.

Preferred input variables include, but are not limited to, viruses,viral particles, liposomes, nanoparticles, biodegradable polymers,radiolabeled particles, radiolabeled biomolecules, toxin-conjugatedparticles, toxin-conjugated biomolecules, and particles or biomoleculesconjugated with stabilizing agents. A “stabilizing agent” is an agentused to stabilize drugs and provide a controlled release. Such agentsinclude albumin, polyethyleneglycol, poly(ethylene-co-vinyl acetate),and poly(lactide-co-glycolide).

A plurality of assays may be run in parallel with different inputvariable concentrations to obtain a differential response to the variousconcentrations. As known in the art, determining the effectiveconcentration of an agent typically uses a range of concentrationsresulting from 1:10, or other log scale, dilutions. The concentrationsmay be further refined with a second series of dilutions, if necessary.Typically, one of these concentrations serves as a negative control,i.e., at zero concentration or below the level of detection.

Input variables of interest encompass numerous chemical classes, thoughfrequently they are organic molecules. A preferred embodiment is the useof the methods of the invention to screen samples for toxicity, e.g.,environmental samples or drug. Candidate agents may comprise functionalgroups necessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The candidate agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Candidateagents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

Included are pharmacologically active drugs and genetically activemolecules. Compounds of interest include chemotherapeutic agents,anti-inflammatory agents, hormones or hormone antagonists, ion channelmodifiers, and neuroactive agents. Exemplary of pharmaceutical agentssuitable for this invention are those described in “The PharmacologicalBasis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y.,(1996), Ninth edition, under the sections: Drugs Acting at Synaptic andNeuroeffector Junctional Sites; Drugs Acting on the Central NervousSystem; Autacoids: Drug Therapy of Inflammation; Water, Salts and Ions;Drugs Affecting Renal Function and Electrolyte Metabolism;Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function; DrugsAffecting Uterine Motility; Chemotherapy of Parasitic Infections;Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases;Drugs Used for Immunosuppression; Drugs Acting on Blood-Forming Organs;Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology,all incorporated-herein by reference. Also included are toxins, andbiological and chemical warfare agents, for example see Somani, S. M.(Ed.), “Chemical Warfare Agents,” Academic Press, New York, 1992).

Test compounds include all of the classes of molecules described above,and may further comprise samples of unknown content. While many sampleswill comprise compounds in solution, solid samples that can be dissolvedin a suitable solvent may also be assayed. Samples of interest includeenvironmental samples, e.g., ground water, sea water, or mining waste;biological samples, e.g., lysates prepared from crops or tissue samples;manufacturing samples, e.g., time course during preparation ofpharmaceuticals; as well as libraries of compounds prepared foranalysis; and the like. Samples of interest include compounds beingassessed for potential therapeutic value, e.g., drug candidates fromplant or fungal cells.

The term “samples” also includes the fluids described above to whichadditional components have been added, for example, components thataffect the ionic strength, pH, or total protein concentration. Inaddition, the samples may be treated to achieve at least partialfractionation or concentration. Biological samples may be stored if careis taken to reduce degradation of the compound, e.g., under nitrogen,frozen, or a combination thereof. The volume of sample used issufficient to allow for measurable detection, usually from about 0.1 μlto 1 ml of a biological sample is sufficient.

Compounds and candidate agents are obtained from a wide variety ofsources including libraries of synthetic or natural compounds. Forexample, numerous means are available for random and directed synthesisof a wide variety of organic compounds and biomolecules, includingexpression of randomized oligonucleotides and oligopeptides.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or readily produced.Additionally, naturally or synthetically produced libraries andcompounds are readily modified through conventional chemical, physicaland biochemical means, and may be used to produce combinatoriallibraries. Known pharmacological agents may be subjected to directed orrandom chemical modifications, such as acylation, alkylation,esterification, amidification to produce structural analogs.

Output variables: Output variables are quantifiable elements of cells,particularly elements that can be accurately measured in a highthroughput system. An output can be any cell component or cell productincluding, e.g., viability, respiration, metabolism, cell surfacedeterminant, receptor, protein or conformational or posttranslationalmodification thereof, lipid, carbohydrate, organic or inorganicmolecule, mRNA, DNA, or a portion derived from such a cell component.While most outputs will provide a quantitative readout, in someinstances a semi-quantitative or qualitative result will be obtained.Readouts may include a single determined value, or may include mean,median value or the variance. Characteristically a range of readoutvalues will be obtained for each output. Variability is expected and arange of values for a set of test outputs can be established usingstandard statistical methods.

Various methods can be utilized for quantifying the presence of theselected markers. For measuring the amount of a molecule that ispresent, a convenient method is to label the molecule with a detectablemoiety, which may be fluorescent, luminescent, radioactive, orenzymatically active. Fluorescent and luminescent moieties are readilyavailable for labeling virtually any biomolecule, structure, or celltype. Immunofluorescent moieties can be directed to bind not only tospecific proteins but also specific conformations, cleavage products, orsite modifications like phosphorylation. Individual peptides andproteins can be engineered to autofluoresce, e.g., by expressing them asgreen fluorescent protein chimeras inside cells (for a review, see Joneset al. (1999) Trends Biotechnol. 17(12):477-81).

Output variables may be measured by immunoassay techniques such as,immunohistochemistry, radioimmunoassay (RIA) or enzyme linkedimmunosorbance assay (ELISA) and related non-enzymatic techniques. Thesetechniques utilize specific antibodies as reporter molecules that areparticularly useful due to their high degree of specificity forattaching to a single molecular target. Cell based ELISA or relatednon-enzymatic or fluorescence-based methods enable measurement of cellsurface parameters. Readouts from such assays may be the meanfluorescence associated with individual fluorescent antibody-detectedcell surface molecules or cytokines, or the average fluorescenceintensity, the median fluorescence-intensity, the variance influorescence intensity, or some relationship among these.

Data analysis; The results of screening assays may be compared toresults obtained from reference compounds, concentration curves,controls, etc. The comparison of results is accomplished by the use ofsuitable deduction protocols, AI systems, statistical comparisons, etc.

A database of reference output data can be compiled. These databases mayinclude results from known agents or combinations of agents, as well asreferences from the analysis of cells treated under environmentalconditions in which single or multiple environmental conditions orparameters are removed or specifically altered. A data matrix may begenerated, where each point of the data matrix corresponds to a readoutfrom a output variable, where data for each output may come fromreplicate determinations, e.g., multiple individual cells of the sametype.

The readout may be a mean, average, median or the variance or otherstatistically or mathematically derived value associated with themeasurement. The output readout information may be further refined bydirect comparison with the corresponding reference readout. The absolutevalues obtained for each output under identical conditions will displaya variability that is inherent in live biological systems and alsoreflects individual cellular variability as well as the variabilityinherent between individuals.

Cell Cultures and Cell Culture Devices

The culture devices of the invention comprise a microfluidic network ofchannels segregated into one or more discrete but interconnectedchambers, preferably integrated into a chip format. The specific chambergeometry is designed to provide cellular interactions, liquid flow, andliquid residence parameters that correlate with those found for thecorresponding cells, tissue, or organ systems in vivo.

Optimized chamber geometries can be developed by repeating the procedureof testing parameter values in response to fluid flows and changes indimensions, until the selected values are obtained. Optimization of thesubstrate includes selecting the number of chambers, choosing a chambergeometry that provides the proper cell to volume ratio, selecting achamber size that provides the proper tissue or organ size ratio,choosing the optimal fluid flow rates that provides for the correctliquid residence time, then calculating the cell shear stress based onthese values. If the cell shear stress is over the maximum allowablevalue, new parameter values are selected and the process is repeated.Another embodiment of the CCA device includes where the cells are grownwithin hollow tubes rather than on the bottom and sides of channels orchambers. It has been demonstrated that cells growing in such athree-dimensional tissue construct are more authentic with respect tocertain in vivo tissues (Griffith (1998) PhARMA Biol. Biotech. Conf.,Coronado, Calif., March 15-18).

Three primary design parameters are considered in creating the 3-Dculture device. The first is the residence time that the fluid is incontact with a particular tissue or within a well. The residence timesare chosen to reflect the amount of time blood stays in contact withorgan tissue, represented by a well, in one pass of the circulatorysystem. The second is the radius of the tubes the cells are grown in.For example, the radius of the tubes for replicating liver are within arange of 200-400 μm. It should be noted that if the radius of the tubesgets too large, the cells will essentially see a flat surface and willform a monolayer on the tube.

The third parameter is the proportion of flow that arrives at eachmodule. Adjusting the geometry of the flow channels partitions the flowfrom the chambers. The channels or tubes to each module or chamber aretypically of different lengths to equilibrate the pressure drops andbalance the flow. After the fluid leaves the other tissues, it can bere-circulated by a pump. The flow rate through the tubes was calculatedfrom the tube dimensions and the residence time. Given a flow rate, theshear stress on the cells was calculated to ensure that the value didnot exceed the cells' stress limit. The very short residence timerequired in the lung tissue makes it impossible to use a well and tubeapproach for this organ. The shear stress is too high and therefore, thelung tissue section remains flow-over with a lung tissue monolayer.

Since the system of the present invention is interactive (i.e., thecomputer not only senses but also controls the conditions within thetest), corrections can be dynamically instituted into the system andappropriately noted and documented for apprising researchers of thedynamics of the test being run.

Data gathering by the computer consists of the collection of datarequired for continuous in-line monitoring of test chemical effluentfrom each compartment. Sensors, preferably of the flow-through type, aredisposed in-line with the outflow from each compartment, to thus detect,analyze and provide quantitative data regarding the test chemicaleffluent from each compartment.

Microprocessors can also serve to compute a physiologically-basedpharmacokinetic (PBPK) model for a particular test chemical. Thesecalculations may serve as the basis for setting the flow rates amongcompartments and excretion rates for the test chemical from the system.However, they may also serve as a theoretical estimate for the testchemical. At the conclusion of the experiment, predictions concerningthe concentrations of test chemicals and metabolites made by the PBPKdetermination can be compared to the sensor data. Hard copy outputcompares the PBPK model with experimental results.

Several prototype CCA systems have been constructed and tested. FIG. 17Adepicts a “first generation” three compartment device. The dimensionswere as follows: wafer was 2 cm×2 cm; lung chamber had 20 channels (5 mmlong) 40 μm×20 μm (w×d); liver chamber had 2 channels (100 mm long) 100μm×20 μm (w×d). The first step in using this device is to inject thefluid using a syringe pump until all the channels filled up. Second, aperistaltic pump is used to recirculate the fluid. FIG. 17B shows across-sectional view of the device, demonstrating the fluidics of thesystem. It was found that 400 μm thick elastomer gave a better seal, andthat plexiglass and gel-loading tips are much less fragile than othermaterials. This device had problems with a high pressure drop and leaksoccurred at 90° bends.

Cell attachment studies were performed using this “first generation”device. L2 cells were placed in the lung chamber and H4IIE cells wereplaced in the liver chamber. Poly-D-lysine was adsorbed to the surfaceof the chambers to promote attachment of the cells within the channels.Unfortunately, cells attached outside the trenches, so differentsubstrates were tested and surfaces were modified.

FIG. 18A depicts a “second generation” device. The dimensions were asfollows: chip was 2 cm×2 cm; etching is 20 μm deep; lung chamber was 2mm×2 mm (w×l); liver chamber was 7.5 mm×10 mm (w×l). The lung chambercontained 5 μm tall ridges to increase cell attachment (FIG. 18B), andthe liver chamber contained 20 μm tall pillars to simulate percolation(FIG. 18C).

FIG. 19 depicts a “third generation” device. The dimensions were asfollows: chip was 2 cm×2 cm; lung chamber was 2 mm×2 mm (w×l); liverchamber was 3.7 mm×3.8 mm (w×l); and the “other tissue” chamber was 7mm×7 mm (w×l). Fluid was split from the lung chamber, with 20% going tothe liver and 80% to the other tissue chamber. Portions of the chambers(dashed) are 100 μm deep to reduce pressure drops, and other portions(solid) are 20 μm deep to give realistic liquid-cell ratios.

FIG. 20 is a flow diagram for a five compartment PBPK model CCA. Thisdevice adds chambers for fat cells, a chamber for slowly perfused fluidand for rapidly perfused fluid. Such a device can be used forbioaccumulation studies, cytotoxicity studies and metabolic activities.Other devices can be developed with various permutations. For example, adiaphragm pump with gas exchange can be added, or an online biosensor,or a microelectromechanical (MEM) pump, or a biosensor and electronicinterface. A device can be developed to mimic oral delivery of apharmaceutical. Alternatively, a device can be developed to mimic theblood-brain barrier.

Fabrication

The cell culture device typically comprises an aggregation of separateelements, e.g., chambers, channels, inlet, or outlets, which whenappropriately mated or joined together, form the culture device of theinvention. Preferably the elements are provided in an integrated,“chip-based” format.

The fluidics of a device are appropriately scaled for the size of thedevice. In a chip-based format, the fluidic connections are“microfluidic,” such a system contains a fluidic element, such as apassage, chamber or conduit that has at least one internalcross-sectional dimension, e.g., depth or width, of between about 0.1 μmand 500 μm. In the devices of the present invention, the channelsbetween chambers typically include at least one microscale channel.

Typically, microfluidic devices comprise a top portion, a bottomportion, and an interior portion, wherein the interior portionsubstantially defines the channels and chambers of the device. Inpreferred aspects, the bottom portion will comprise a solid substratethat is substantially planar in structure, and which has at least onesubstantially flat upper surface. A variety of substrate materials maybe employed as the bottom portion. Typically, because the devices aremicrofabricated, substrate materials will generally be selected basedupon their compatibility with known microfabrication techniques, e.g.,photolithography, thin-film deposition, wet chemical etching, reactiveion etching, inductively coupled plasma deep silicon etching, laserablation, air abrasion techniques, injection molding, embossing, andother techniques.

The substrate materials of the present invention comprise polymericmaterials, e.g., plastics, such as polystyrene, polymethylmethacrylate(PMMA), polycarbonate, polytetrafluoroethylene (TEFLON™),polyvinylchloride (PVC), polydimethylsiloxane (PDMS), polysulfone, andthe like. Such substrates are readily manufactured from microfabricatedmasters, using well known molding techniques, such as injection molding,embossing or stamping, or by polymerizing the polymeric precursormaterial within the mold. Such polymeric substrate materials arepreferred for their ease of manufacture, low cost and disposability, aswell as their general inertness to most extreme reaction conditions.These polymeric materials may include treated surfaces, e.g.,derivatized or coated surfaces, to enhance their utility in the system,e.g., provide enhanced fluid direction, cellular attachment or cellularsegregation.

The channels and/or chambers of the microfluidic devices are typicallyfabricated into the upper surface of the substrate, or bottom portion,using the above described microfabrication techniques, as microscalegrooves or indentations. The lower surface of the top portion of themicrofluidic device, which top portion typically comprises a secondplanar substrate, is then overlaid upon and bonded to the surface of thebottom substrate, sealing the channels and/or chambers (the interiorportion) of the device at the interface of these two components. Bondingof the top portion to the bottom portion may be carried out using avariety of known methods, depending upon the nature of the substratematerial. For example, in the case of glass substrates, thermal bondingtechniques may be used that employ elevated temperatures and pressure tobond the top portion of the device to the bottom portion. Polymericsubstrates may be bonded using similar techniques, except that thetemperatures used are generally lower to prevent excessive melting ofthe substrate material. Alternative methods may also be used to bondpolymeric parts of the device together, including acoustic weldingtechniques, or the use of adhesives, e.g., UV curable adhesives, and thelike.

The device will generally comprise a pump, such as a low flow rateperistaltic pump. A small bore flexible tubing would be attached to theoutlet of the device, passing through the peristaltic pump and attachedto the inlet of the device, thus forming a closed loop system. The pumpgenerally operates at flow rates on the order of 1 μL/min. The pumpsystem can be any fluid pump device, such as a diaphragm, and can beeither integral to the CCA device (chip-based system) or a separatecomponent as described above.

The device can be connected to or interfaced with a processor, whichstores and/or analyzes the signal from each the biosensors. Theprocessor in turn forwards the data to computer memory (either hard diskor RAM) from where it can be used by a software program to furtheranalyze, print and/or display the results.

Description of Exemplary Embodiments

In the following detailed description of specific embodiments, referenceis made to the accompanying drawings, which form a part hereof, and inwhich are shown by way of illustration specific embodiments in which theinvention may be practiced. It is to be understood that otherembodiments may be utilized and structural changes may be made withoutdeparting from the scope of the present invention.

FIG. 1 is a block diagram of an in vitro system in accordance with thepresent invention. Lung cell simulating chamber 102 receives oxygenatedculture medium from gas exchange device 103. Such oxygenated medium isobtained by contacting culture medium with oxygen-containing gas so thatthe culture medium absorbs oxygen-containing gas and desorbs carbondioxide-containing gas. The culture medium exiting lung cell simulatingchamber 102 is analogous to arterial blood 106 in mammals. Theoxygen-containing culture medium constituting arterial blood 106 is thensupplied to liver simulating chamber 108, other tissue simulatingchamber 110, fat simulating chamber 112, and kidney simulating chamber114. The culture medium departing from liver simulating chamber 108,other tissue simulating chamber 110, fat simulating chamber 112, andkidney simulating chamber 114 is analogous to venous blood 104 inmammals. As shown in FIG. 1, the culture medium corresponding to venousblood 104 is returned to lung cell simulating chamber 102. The system ofthe present invention also includes gut simulating chamber 116 andperitoneal cavity simulating chamber 118, both of which constitute sitesfor introduction of test compounds. As in mammals, waste liquid 115 iswithdrawn from kidney simulating chamber 114.

FIG. 2 is a simplified schematic view of one embodiment of the system200 of the present invention. The system 200 includes a lung cellculture chamber 210, a liver cell culture chamber 212, a fat cellculture chamber 213, an other tissues chamber 214, and a gas exchangechamber 250. The chambers 210, 212, 213, 214, and 250 are formed on asubstrate of silicon that is commonly referred to as a chip 230. Itshould be noted that more than four cell culture chambers may be housedor formed on a single chip 230. A fluid path 240 connects the chambers210, 212, 213, 214, and 250.

The chambers have an inlet 211 and an outlet 215. The inlet 211 islocated at one end of the gas exchange chamber 250. The outlet 215 islocated at one end of the liver cell culture chamber 212. The chambers210, 212, 213, 214, and 250 and the fluid path 240 are locatedsubstantially between the inlet 211 and the outlet 215. The systemincludes a pump 260 for circulating the fluid in the system 200. Amicrotube 270 connects between the outlet 215 and the inlet side of thepump 260. A microtube 271 connects the outlet side of the pump 260 tothe inlet 211. The cell culture chambers 210, 212, 213, 214 the gasexchange chamber 250, the fluid path 240, and the pump 260 form thesystem 200. The system may include additional cell culture chambers. Onecommon cell culture chamber added is one simulating kidney.

FIG. 3 is a schematic of another embodiment of the invention. In FIG. 3a first signal path 310, a second signal path 320, and a third signalpath 330 are provided on the chip 230. Signals for monitoring variousaspects of each cell culture system 200 can be taken from the chip 230and at specific locations on the chip 230 and moved to outputs off thechip 230. One example, the signal paths 310, 320, 330 on the chip 230are integrated buried waveguides. The chip 230, in such an embodiment,could be made of silicon, glass or a polymer. The waveguide 310, 320,330 would carry light to the edge of the chip where a transducer 312,322, 332 would be located to transform the light signal to an electricalsignal. The cells within the system 200 could then be monitored forfluorescence, luminescence, or absorption or all these properties tointerrogate and monitor the cells within the system 200. Checkingfluorescence requires a light source. The light source is used tointerrogate the molecule and the signal carrier, such as a waveguide310, 320, 330 or a fiber optic captures the signal and sends it off thechip 230. The signal carrier, 310, 320, 330 would direct light to aphotodetector near the end of the signal carrying portion of the chip310, 320, 330.

FIG. 4 is a schematic view of another embodiment of the system 200 ofthe present invention. In this embodiment, biosensors 410, 420, 430,440, 450, and 460 are positioned on the chip upstream and downstream ofeach of the cell culture chambers of the chip 230. The biosensors 410,420, 430, 440, 450, 460 monitor the oxygen, carbon dioxide, and/or pH ofthe medium. These sensors allow monitoring of the system 200 andadjustment of gas levels as needed to maintain a healthy environment. Inaddition, if positioned just upstream and downstream of each cellcompartment, biosensors provide useful information on cellularmetabolism and viability.

FIGS. 5A through 5G show steps used to fabricate a polymer-baseddisposable chip 230. A silicon wafer 20 is spin coated with a thin layerof photoresist 21 (FIG. 5A). The photoresist 21 is exposed to UV light22 through a photomask 23 containing the desired features (FIG. 5B). TheUV exposed photoresist 21 is developed away in an appropriate solventthus exposing the silicon 20 (FIG. 5C). The silicon 20 is etched to adesired depth using an inductively coupled plasma etching system (FIG.5D). The remaining photoresist is removed with an appropriate solvent(FIG. 5E). A very thin gold (or Ti) plating base 24 is deposited on thesilicon substrate 20 creating a template for the electroplating processas shown in FIG; 5E. The sample is immersed in a nickel sulfamate typeplating bath and nickel 25 is electroplated onto the silicon template 20until the nickel thickness is sufficient, with the gold acting as aconducting layer. The nickel master grows off the gold layer, and thegold becomes a part of the nickel master. This forms Ni features 25,shown in FIG. 5F. The plating rate, which is a function of platingcurrent, template diameter and template thickness, is calibrated forabout 45 nm/min. After fabrication, the features 25 are examined using amicroscope to verify the feature dimensions. The resulting nickelfeatures 25 must be uniform and have the desired shape. The nickelmaster 25 and the polymer substrate 26 are heated to just above theglass transition temperature of the polymer. The nickel master 25 andpolymer 26 are brought into contact and the features of the nickelmaster 25 are embossed into the polymer substrate 26. The nickel master25 is removed thus producing a polymer 26 containing the identicalfeatures of the original silicon wafer 20 (FIG. 5G).

FIG. 6 is a schematic view of a third embodiment of the system 200 ofthe present invention. In this embodiment, biosensors 600, 602, 604 arepositioned about the periphery of the chip 230. The biosensors 600, 602,604 are used to further monitor the status of the cells of the system200 created on the chip 230. Advantageously, by positioning thebiosensors 600, 602, 604 about the periphery of the chip 230, the chip230 could be made to be disposable with the least amount of cost. Inother words, the biosensors 600, 602, 604 would not have to be thrownaway with the chip 230. It should be noted that biosensors 600, 602, 604may also be provided on board the disposable chip 230. This particularoption would not be as cost effective since the biosensors 600, 602, 604disposing the chip 230 also results in throwing away the biosensors 600,602, 604. It is more cost effective when the biosensors 600, 602, 604are positioned off the chip 230 since the biosensors 600, 602, 604 arereused rather than disposed of after each use. Each of the biosensors600, 602, 604 is connected to the inputs of a computer 620.

FIG. 7 is a schematic further detailing the computer 620. The computer620 monitors and regulates operations of the system 200 of each chip230. Computer 620 includes a microprocessor provided with input/outputinterface 700 and internal register/cache memory 702. As shown,microprocessor 798 interfaces to keyboard 704 through connection 716, tonon-volatile storage memory 706, general purpose memory 708, and look-uptables 710 through connector 718, and to printer/plotter recorder 712and display 714 through connector 720.

Non-volatile storage memory 706 may be in the form of a CD writeablememory, a magnetic tape memory, disk drive, or the like. Look-up tables710 may physically comprise a portion of general purpose memory 708 thatis set aside for storage of a set of mass balance equations applicableto various substances to be modeled in the system. These equationsrepresent physiologically-based pharmacokinetic models for variousbiological/chemical substances in systems internal register/cache memory702 and general purpose memory 708 contain a system program in the formof a plurality of program instructions and special data forautomatically controlling virtually every function in the system 200 ofeach chip 230. The computer can also control and regulate the pump 260associated with the system 200.

Fluid flow may also be provided as inputs to microprocessor 798 throughinput/output interface 700 from flow meters. This permits precisecontrol over fluid flow rates within the system by adjustment of programcommands that are transmitted to pumps 260 through pump control lines,respectively. For example, the flow rates may be set to 9.5 μL/min. inconduit 58, 2.5 μL/min. through flow meter 66, 7 μL/min. through flowmeter 78, and 2.5 μL/min. in conduit 70. The temperature of culturemedium in reservoir 50 may also be regulated by microprocessor 798,which receives, through input/output interface 700 and temperatureindicator line 728, temperature measurements from temperature probe 792.In response to these signals, heater coil 790 is turned on and off bymicroprocessor 798 through input/output interface 700 and heater coilcontrol line 730.

Biological and toxicological reactions/changes in cell culture chambers210 and 212 are detected by sensors 600, 602 and 604, respectively, andcommunicated to microprocessor 798 through control lines as well asinput/output interface 700. The sensors can be designed to representtest results in terms of specific values or ranges of wavelengths torepresent test results.

Microprocessor 798 is also quite easily adaptable to include a programto provide the researcher with interactive control via keyboard 704.This permits, for example, directing the computer to specifically checkon the conditions of any of the culture compartments at any given time.

A further option provided by the present invention is the ability torecall previously stored test results for similar experiments byrecalling information from the CD/tape memory 706. Thus, memory 706 maybe preprogrammed to hold historical data taken from publishedinformation, data gathered from previously run tests conducted with thesystem of the present invention or data derived from theoreticalcalculations. The provision of the CD/tape memory also permits thesystem to be used as an information researching tool. It can, forexample, obtain the research data pertaining to a particular testchemical, or to a particular culture line, based on selectioninformation inputted into microprocessor 798 via keyboard 704. Byincluding or developing a large library of information in memory 706,researchers will be able to configure and plan test runs moreintelligently.

FIG. 8 is a schematic showing that more than one chip 230 can be housedwithin a single housing 800. The housing 800 can be an environmentalchamber that maintains the same conditions for each of the chips 230within the housing. The housing 800 includes a plurality of chiplocations 810, 812, 814, 816. The outputs from each chip 230 or chiplocation 810, 812, 814, 816 is input to a computer 620. The computer 620is then able to monitor the systems 200 from multiple chips 230 in realtime.

FIG. 9 is a schematic showing that a test may include sets of chips 230in different housings 800, 900. The outputs of each of the chips 230 canbe monitored for changes in the environment, such as when temperature isslightly elevated, or the like. It is further contemplated that each ofthe chips in one housing may have the same cell culture thereon or thatthe chips 230 in the housing 800 may have chips interconnected to oneanother to form different portions of a mammal or interdependent organswithin a housing.

The chips 230 discussed with respect to FIGS. 2-4 and 6-9 use twodimensional cell culture chambers 210, 212, 213, 214. Since threedimensional tissue culture constructs may be more authentic in theirmetabolism, yet another of the chip 1000 addresses the inclusion ofthree dimensional constructs. The following describes the creation of amicroscale cell culture analogous device (“CCA”), which incorporatesthree dimensional tissues in a modular format. The CCA device or chip1000 incorporates a flow over approach for lung cell chambers and aflow-through approach for other organs. The flow-through approach to CCAdesign is further discussed below.

FIG. 10 shows a schematic and flow regime for a chip 1000. The chip 1000includes four wells or tissue modules. The chip 1000 includes a lungwell 1010, a liver well 1020, a fat well 1030, and a slowly perfusedwell 1040, and a rapidly perfused well 1050. Tubes are used to circulatea fluid through the chip 1000. A pump 1060 moves the fluid through thetubes. The lung well 1010 initially receives all of the flow. After thelung 1010, the fluid will partition into the four tissue modules. Theliver module will get 25% of the flow, the fat module 9%, the slowlyperfused module 15% and the rapidly perfiused section 51%. Adjusting thegeometry of the flow channels will partition the flow from the lung well1010. The channels to each module will be of different lengths toequilibrate the pressure drops and balance the flow. After the fluidleaves the other tissues, it will be re-circulated back into the lungcompartment via the pump 1060. Each of the wells or tissue modules 1020,1030, 1040, 1050 holds tissue. The tissue is held in microscale tubes1022, 1032, 1042, 1052 within the wells 1020, 1030, 1040, 1050. As shownin FIG. 10, there is only one microscale tube 1022, 1032, 1042, 1052 perwell 1020, 1030, 1040, 1050. It should be noted that a plurality ofmicrotubes may be placed in a well.

In operation, there are two methods that allow three dimensional tissueto be incorporated into a CCA device or chip 1000. Both methods involvethe flow of inoculated medium through microscale tubes of polystyrene orglass. The cells under test adhere to the inside of the tubes andaggregate into three dimensional tissue. The tubes are collected,bundled and placed into wells on a chip 1000. Each well becomes an organmodule that the aqueous drug will flow through to contact the tissue.

The first method to allow incorporation of three dimensional tissueinvolves a flow-through reactor strategy. Openings are formed in asilicon wafer and channeled medium is then passed through the openings.The silicon on the inside surface of the openings provided a scaffoldfor the cells and they aggregated into three dimensional tissue. Toapply this technique to a polymer CCA 1000, the polymer tubes can eitherbe treated with an adhesion protein or the cells can be cultured inserum-added medium. Both serum and an adhesion protein allow the cellsto stick to the inside surface of the tube.

The second method involves culturing the cells in a HARV microgravityreactor. By scaffolding the tubes in the center of the rotating reactor,or by introducing free-floating tubes into the culture medium, the cellsform three dimensional aggregates in some of the tubes. Due to theheightened activity of cells grown in microgravity, these tissueconstructs have superior function compared to two dimensional tissue orthe tissue formed in the method above. The tubes with tissue inside ofthem can be separated according to weight or density and placed on thedevice.

FIG. 11 is a partially exploded isometric view of a cell culture analogdevice 1100 that incorporates chip 1000. The chip 1000 includes a lungcell culture area 1010 and a plurality of wells that are connected tothe lung cell culture area 1010. The wells include a liver tissue well1020, a fat tissue well 1030, a slowly perfused well 1040, and a rapidlyperfused well 1050. Microscale tubes containing the various tissues fitwithin the well 1020, 1030, 1040, and 1050. Each well includes an outputto an elastomeric bottom 1110 that is attached to the chip 1000. Theelastomer 1110 is part of a pump. An actuator 1120 presses against theelastomer to produce a pumping action to move the fluid of the system1100 or to circulate the fluid of the system 1100 from the wells back tothe lung tissue module 1010 via a return line 1130. A glass layer isplaced over the top of the chip to cover the lung tissue module 1010 andthe various wells 1020, 1030, 1040, and 1050. It should be noted thatthe channels 1021, 1031, 1041, and 1051 are dimensioned to producecertain flow rates through the various wells 1020, 1030, 1040, and 1050.Rather than adjust the length and width of the various channels 1021,1031, 1041, 1051 it is contemplated that other flow restrictors can beplaced along the channel in order to provide for variability within theflow rates to the various wells 1020, 1030, 1040, and 1050. The glasstop 1140 can be replaced with a membrane that flexes and plungerball-type valves can be added so that the flows in the channels 1021,1031, 1041, and 1051 can be regulated by other than the dimensions ofthe channel.

The chip 1100 can be made out of silicon but is more cost effective tomake the chip 1000 out of polystyrene or some other suitable plastic.Each chip is first formed in silicon by conventional means. A nickelmaster is then formed from the silicon. In other words, the chip 1000 ismanufactured by replica molding polystyrene and silicone elastomer onsilicon and nickel masters. Of course, the first step in the manufactureof a polymer chip is to produce the chip on a silicon wafer. Initially,a layer of photoresist 1210 is placed on a silicon wafer 1200. A mask isplaced over the photoresist 1210. The mask contains the pattern of alung tissue culture area 1010. The mask allows UV light to pass to thephotoresist to expose just the portion corresponding to the lung area1010. The photoresist is then developed to produce an opening 1211,which corresponds to the lung tissue culture area 1010. The siliconwafer with the photoresist is then etched to produce the lung opening1010 within the silicon wafer 1200. The photoresist 1210 is then removedfrom the silicon wafer 1200 leaving the silicon wafer with the lung well1010. Another layer of photoresist 1220 is then placed onto the wafer1200. A mask is placed over the wafer. The mask allows for exposure ofthe various wells or fluid channels including 1021, 1031, 1041, and1051, which are used to connect the lung well 1010 with the variouswells 1020, 1030, 1040, and 1050. The mask exposes the photoresist inthe area of the fluid channel. The photoresist is then developed toremove the exposed photoresist corresponding to the fluid flow channels.The exposed area is then etched to a desired depth. Afterwards, theremaining photoresist 1220 is removed leaving a silicon wafer 1200 witha lung well 1010 and other wells 1020, 1030, 1040, and 1050. The nextstep is to apply yet a third layer of photoresist 1230. A mask is placedover the photoresist and the mask has openings corresponding to thevarious wells 1020, 1030, 1040, and 1050. The photoresist is masked andexposed to UV light to produce openings corresponding to the variouswells. The photoresist is developed leaving the exposed silicon areasfor wells 1020, 1030, 1040, and 1050. The chip and the photoresist 1230are then etched to produce the wells 1020, 1030, 1040, and 1050. Theopenings corresponding to the tissue modules 1020, 1030, 1040, 1050 isetched with plasma to a depth of approximately 750 micrometers. Theopenings are then wet etched another 250 micrometers with KOH to form atapered end. The KOH will etch silicon along its crystallographic planeat an angle of 54.7 degrees. The photoresist is then removed and asilicon wafer has been formed from which the nickel master can be made.

Nickel is electroplated onto the silicon chip to create a nickel master1250. The nickel master is then used to cast or emboss the polymersubstrate 1000. For replica molding, the polymer is melted orsolubilized in an appropriate solvent and poured onto the nickel master1250 and solidifies in the same shape as the initial silicon chip. Forembossing, refer to FIG. 5. The polymer chip 1000 is then mounted on asilicone elastomer trough 1110. The polymer and silicone areself-sealing so the layers will form a single unit. A pneumatic actuator1120 is put below the chip to pump fluid collected from the varioustissue modules 1020, 1030, 1040, 1050. Every second, the trough willfill up with 0.032 microliters of fluid. The actuator will then push upon the silicone and cause the fluid to escape through the microtubesback to the lung compartment 1010. The elastomeric trough 1110 and theactuator 1120 form the pump 260 (shown in FIG. 12). The elastomer-coatedpolymethylmethacrylate (PLEXIGLAS™) 1140 is then sealed to the top ofthe wafer or chip 1000.

To balance the pressure pull created as the silicone fills up withliquid, the polymethylmethacrylate (PLEXIGLAS™) over the lung cellcompartment 1010 is removed and replaced with a silicone membrane. Thismembrane rises and falls in response to the action of the silicone pumpand keeps the pressure in the device balanced. The various microscaletubes are placed into the wells prior to placing the elastomer-coatedpolymethylmethacrylate (PLEXIGLAS™) over the chip 1000. A machine forhandling the microtubes includes an adhesive arm that lowers andcollects a specific number of tissue-laden tubes. The machine transportsthe tubes to the device and tightly packs the tubes into the respectivemodule wells 1020, 1030, 1040, 1050. The tight packing allows the forceof friction to keep the tubes in place regardless of any agitation tothe cell culture analog device. This minimizes leakage of fluid flowaround the tubes in the respective wells 1020, 1030, 1040, 1050. Evenwith a tight fit, approximately 5-10% of the fluid flow circumvents thetubes and flows directly to the silicone base or elastomer trough 1110.

FIG. 13 shows the elastomer trough. The elastomer trough is a piece ofsilicone elastomer with an essentially rectangular opening therein. Therectangular opening acts as a fluid reservoir for the fluids coming fromthe wells 1020, 1030, 1040, and 1050. The elastomer trough 1110 has anopening in one side designated by reference numeral 1300. The returnline 1130 has one end that attaches to the opening 1300 in the elastomertrough 1110 and another end that attaches to the lung well 1010 of thechip 1000.

In yet another embodiment, the elastomer trough 1110 is replaced with asilicone elastomer pump 1400, which is shown in FIG. 14. The siliconeelastomer pump 1400 is designed to more accurately reproduce thecirculatory system flow on the chip 1000 and throughout the systemdepicted by reference numeral 1100. The pump 1400 includes a firstpulmonary chamber 1410 and a second system chamber 1412, which areactuated by separate actuators 1420 and 1422. With the multiple chambers1410 and 1412 a more physiologically realistic pumping pattern iscreated with the multi-trough elastomeric base on the bottom of the chip1000. By creating the multiple chambers 1410 and 1412 in the siliconeelastomer trough 1400 by having actuators that push up on the section ofthe base at specific time intervals, the pumping action of a heart isreplicated.

FIG. 28A is a block-diagram view illustrating a system for controlling amicroscale culture device, according to one embodiment of the presentinvention. In this embodiment, the system 2800 includes a firstmicroscale culture device 2806 coupled to a control instrument 2802. Thefirst microscale culture device 2806 includes a number of microscalechambers (2808, 2810, 2812, and 2814) with geometries that simulate anumber of in vivo interactions with a culture medium, wherein eachchamber includes an inlet and an outlet for flow of the culture medium,and a microfluidic channel interconnecting the chambers. The controlinstrument 2802 includes a computer 2804 to acquire data from, andcontrol pharmacokinetic parameters of, the first microscale culturedevice 2806.

In another embodiment, the first microscale culture device 2806 isformed on a computerized chip. The first microscale culture device 2806further includes one or more sensors coupled to the control instrument2802 for measuring physiological events in the chambers. The sensorsinclude one or more biosensors that monitor the oxygen, carbon dioxide,or pH of the culture medium. The control instrument 2802 holds the firstmicroscale culture device 2806, and seals a top of the first microscaleculture device 2806 to establish the microfluidic channel. The controlinstrument 2802 provides the microfluid interconnects, so thatmicrofluid flows into and out of the device. In another implementation,the computer 2804 controls a pharmacokinetic parameter selected from agroup consisting of group pump speed, temperature, length of experiment,and frequency of data acquisition of the first microscale culture device2806. In one implementation, the computer 2804 provides a set-up screenso that an operator may also manually specify pump speed, devicetemperature, length of experiment, and frequency of data acquisition(e.g., every fifteen minutes). In another implementation, the computer2804 controls a pharmacokinetic parameter selected from a groupconsisting of flow rate, chamber geometry, and number of cells in thefirst microscale culture device 2806. In this implementation, the system2800 provides more rapid and more sensitive responses as compared towhole animal studies and traditional tissue culture studies. Bycontrolling parameters, the system 2800 is no longerphysiologically-based. In another implementation, the computer 2804further controls one or more pumps in the first microscale culturedevice 2806 to create culture medium residence times in the chambers(2808, 2810, 2812, and 2814) comparable to those encountered in theliving body. In another implementation, the computer 2804 furthercontrols one or more valves distributed along the microfluidic channelin a manner that is consistent with a pharmacokinetic parameter valueassociated with a simulated part of a living body.

In another embodiment, the system 2800 further includes a secondmicroscale culture device having a number of microscale chambers withgeometries that simulate a number of in vivo interactions with a culturemedium, wherein each chamber includes an inlet and an outlet for flow ofthe culture medium, and a microfluidic channel interconnecting thechambers. The control instrument 2802 is coupled to the secondmicroscale culture device.

FIG. 28B is a block-diagram view illustrating another embodiment of asystem for controlling a microscale culture device. In this embodiment,the system 2816 includes the first microscale culture device 2806coupled to a control instrument 2818. The control instrument 2818includes the computer 2804, a pump 2820 to control circulation ofmicrofluid in the microfluidic channel of the first microscale culturedevice 2806, a heating element 2822 to control the temperature of thefirst microscale culture device 2806, a light source 2824, and aphotodetector 2826 to detect fluorescent emissions from cellcompartments within the first microscale culture device 2806. In oneimplementation, the computer 2804 records data for fluorescent intensityusing a measuring instrument of a type that is selected from a groupconsisting of colorimetric, fluorometric, luminescent, and radiometric.In another implementation, the heating element 2822 maintains the firstmicroscale culture device 2806 at a temperature of thirty-seven degreesCelsius.

FIG. 29 is a flow-diagram view illustrating a computerized method fordynamically controlling a microscale culture device, according to oneembodiment of the present invention. In this embodiment, thecomputerized method 2900 includes blocks 2902, 2904, 2906, and 2908.Block 2902 includes analyzing data from a number of sensors to measurephysiological events in a number of chambers of the microscale culturedevice. Block 2904 includes regulating fluid flow rates of a culturemedium in the chambers of the microscale culture device. Block 2906includes detecting biological or toxicological reactions in the chambersof the microscale culture device. Upon such detection, block 2908includes changing one or more pharmacokinetic parameters of themicroscale culture device.

In one embodiment, block 2906 (i.e., the detecting) includes detecting achange in dimension of a cell compartment of the microscale culturedevice. In one implementation, block 2908 (i.e., the changing) includeschanging a pharmacokinetic parameter selected from a group consisting ofinteractions between cells, liquid residence time, liquid to cellratios, metabolism by cells, and shear stress in the microscale culturedevice. In another implementation, block 2908 includes changing apharmacokinetic parameter selected from a group consisting of flow rate,chamber geometry, and number of cells in the microscale culture device.

In another embodiment, the computerized method 2900 further includesoptimizing chamber geometry within the microscale culture device,wherein the optimizing includes selecting a quantity of chambers,choosing a chamber geometry that provides a proper tissue or organ sizeratio, choosing an optimal fluid flow rate that provides a proper liquidresidence time, and calculating a cell shear stress.

In another embodiment, the computerized method 2900 further includesregulating a temperature of the culture medium. In yet anotherembodiment, the computerized method 2900 further includes detectingfluorescent emissions from a cell compartment of the microscale culturedevice.

In another embodiment, a computer-readable medium includescomputer-executable instructions stored thereon to perform the variousembodiments of the computerized method described above. In oneimplementation, the computer-readable medium includes a memory or astorage device. In another implementation, the computer-readable mediumincludes a computer data signal embodied in a carrier wave.

FIG. 30 is a block-diagram view illustrating a computer for controllinga microscale culture device, according to one embodiment of the presentinvention. In this embodiment, the computer 3000 includes amicroprocessor 3002, a general memory 3004, a non-volatile storageelement 3006, an input/output interface 3008 that includes an interfaceto a microscale culture device having one or more sensors, and computersoftware. The computer software is executable on the microprocessor 3002to regulate fluid flow rates of a culture medium in a number of chambersin the microscale culture device, detect biological or toxicologicalreactions in the chambers of the microscale culture device, and upondetection, change one or more pharmacokinetic parameters of themicroscale culture device.

In one embodiment, the non-volatile storage element 3006 includeshistorical data taken from published information, data gathered frompreviously run tests, or data derived from theoretical calculations. Thecomputer software regulates the fluid flow rates by transmittingcommands to one or more pumps of the microscale culture device throughpump control lines. In one implementation, the computer software isfurther executable on the microprocessor 3002 to regulate a temperatureof the culture medium. The computer software regulates the temperatureby transmitting commands to a heater coil of the microscale culturedevice through heater coil control lines.

In another embodiment, the computer 3000 further includes a look-uptable memory coupled to the general memory 3004 for storing a set ofmass balance equations that represent physiologically-basedpharmacokinetic models for various biological or chemical substances inthe system, and a cache memory coupled to the microprocessor 3002 forstoring the computer software.

In another embodiment, the input/output interface 3008 further includesa keyboard interface, a display interface, and a printer/plotterrecorder interface. In one implementation, the computer 3000 uses theseinput/output interfaces to connect to keyboard, display, andprinter/plotter recorder peripheral devices.

Experimental

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention.

Efforts have been made to insure accuracy with respect to the numbersused (e.g., amounts, temperature, concentrations) but some experimentalerrors and deviations arise. Unless otherwise indicated, parts are partsby weight, molecular weight is weight average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

Methods

The following methods were used in the experimental process:

Cell culture. Cells were obtained from American Type Culture Collection(Manassas, Va.) and propagated in the recommended complete growth mediumin a tissue culture incubator (95% O₂/5%CO₂). For HepG2 and HepG2/C3Acells, the recommended media is Eagle's Minimum Essential medium (withEarle's balanced salts solution, 2 mM L-glutamine, 1.0 mMsodium-pyruvate; 0.1 mM nonessential amino aids, 1.5 g/L sodiumbicarbonate, and 10% fetal bovine serum) (EMEM). McCoy's 5a medium with1.5 mM L-glutamine, 1.5 g/L sodium bicarbonate and 10% fetal bovineserum is recommended for the HCT116.

Growth curves. Growth curves were determined by plating the cells at aninitial low density in 35 mm dishes. Each day, cells were detached withtrypsin-EDTA and cell number was determined by visually counting thecells using a hemacytometer. Determinations were done in triplicate.

Reverse transcriptase-polymerase chain reaction (RT-PCR). Cells werecultured on glass coverslips treated with collagen, MATRIGEL™, orpoly-lysine as appropriate. HepG2/C3A grown to a ˜90% confluentmonolayer were detached with trypsin-EDTA and pelleted at ˜500 g for 5min. RNA was isolated and purified with RNEASY™ kit (Qiagen) accordingto manufacturer's protocol. Adult human liver total RNA was purchasedfrom Ambion. The quantity and purity (260/280 nm ratio) of isolated RNAwas measured on a BIOPHOTOMETER™ spectrophotometer (Eppendorf). Theisolated RNA was then incubated at 37° C. for 25 min with 2 U of DNase Iand subsequently inactivated with DNase Inactivation Reagent (Ambion).

The RT reaction was performed using a mixture of 5 μg RNA, 10 μM oligodT primers heated to 72° C. for 2 minutes followed by 2 minute on ice.Next, 5 mM DTT, 600 μM dNTP mix, 40 U rRNasin, 200 U SUPERSCRIPT II™ inreverse transcriptase buffer were combined and incubated at 42° C. for 1hour.

2.0 μl of first strand cDNA was used in 50 μl PCR reactions usingcytochrome P450 isoform specific primers (Rodriguez-Antona, C., Jover,R., Gomez-Lechon, M.-J., and Castell, J. V. (2000). Quantitative RT-PCRmeasurement of human cytochrome P-450s: application to drug inductionstudies. Arch. Biochem. Biophys., 376:109-116). PCR conditions were: 94°C. for 4 minutes followed by 28 cycles of 40 seconds at 94° C., 45seconds at 60° C., 50 seconds at 72° C., and a final 4 minutes extensionat 72° C.

PCR products were separated by electrophoresis on a 1.2% agarose gel andvisualized by staining with SYBR Gold and compared to appropriatemolecular weight standards for authenticity. To quantify the amplifiedcDNA, 15 μl of each PCR reaction was diluted with 0.1× Tris-EDTA bufferand stained with PICOGREEN™ (Molecular Probes) at a final concentrationof 1:400. Fluorescence was measured at 480 nm excitation and 520 nmemission. Results were standardized against β-actin and done intriplicate from at least two separate experiments.

Cell viability, death and apoptosis assays. Cell viability and celldeath were determined using trypan blue exclusion or LIVE/DEAD stain(Molecular Probes). Trypan blue (GIBCO), normally excluded from thecytoplasm, identifies cells with compromised membranes by visiblystaining dead or dying cells blue. A 1:1 dilution of a 0.4% (w/v)solution of trvpan blue is added to the recirculating culture medium ofthe chip device at the conclusion of the experiment. This solution waspumped through the chip to waste for 30 minutes at room temperature. Thehousing was removed from the pump and visualized under a reflectingmicroscope (Micromaster, Fisher).

LIVE/DEAD stain is a two-component stain consisting of calcein AM andethidium homodimer. Living cells actively hydrolyze the acetoxymethylester (AM) moiety of calcein AM to produce bright green fluorescence ofcalcein. In contrast, cells that have compromised membrane integrityallow the normally membrane impermeant ethidium homodimer to stain thenucleus of dead or dying cells fluorescent red. The cell permeantnuclear stain, Hoechst 33342 acts as a general stain for all cells.Together with the appropriate filter sets, living cells fluoresce green,dying or dead cells red, and all cells are quantified by a blue nuclearfluorescence. For experiments described herein, trypan blue was used at0.2% (w/v), calcein AM at 1:20,000, propidium iodide at 1:5,000, andHoechst 33342 at 10 μg/ml. Cells were visualized with a M2Biostereofluorescence microscope (Zeiss). All experiments were repeated atleast three times and measurements done in triplicate.

Apoptosis, or programmed cell death, can be monitored using a number ofmethods (Smyth, P. G., Berman, S. A., and Bursztajn, S. (2000). Markersof apoptosis: methods for elucidating the mechanism of apoptotic celldeath from the nervous system. Biotechniques, 32:648-665). Todistinguish apoptosis from necrosis, at least two separate indicators ofapoptosis are required (Wronski, R., Golob, N., and Gryger, E., (2002).Two-color, fluorescence-based microplate assay for apoptosis detection.Biotechniques, 32:666-668. One method, annexin V-FITC binding, relies onthe observation that annexin V binds tightly to phosphatidylserine inthe presence of divalent calcium (Williamson, P., Eijnde, S.v.d., andSchlegel, R. A. (2001). Phosphatidylserine exposure and phagocytosis ofapoptotic cells. In Apoptosis, L. M. Schwartz, and J. D. Ashwell, eds.(San Diego, Academic Press), pp. 339-364). Normally, phosphatidylserineis present on the inner leaflet of cell membranes, but translocates tothe cell membrane early in apoptosis. Apoptotic cells exposed tofluorophore-labeled annexin exhibit distinct membrane staining. With themicroscale chip, annexin V-FITC labeling was visualized directlyon-chip, by first flushing the system with PBS, then recirculatingannexin V-FITC (10 μg/ml in annexin V binding buffer, Clontech) for 30min. Cells were then visualized directly using a FITC filter set.

In contrast to annexin V labeling, the APOPTAG™ kit (Intergen Co.,Massachusetts) uses terminal deoxynucleotidyl transferase to label free3′-OH DNA termini exposed during apoptotic DNA degradation andvisualization using immunofluorescence (Li, X., Traganos, F., Melamed,M. R., and Darzynkiewicz, Z. (1995). Single-step procedure for labelingDNA strand breaks with flourescein-or BODIPY-conjugateddeoxynucleotides: detection of apoptosis and bromodeoxyuridineincorporation. Cytometry 20, 172-180). Although this method is highlyspecific for apoptosis, the procedure cannot be done on-chip due to thefixation and incubation steps. Briefly, microscale chips were run underspecified experimental conditions, the cell chips were removed fromtheir housing units, fixed in 1% paraformaldehyde and processed with theAPOPTAG™ kit using the manufacturer's protocol.

Microscale Chip Fabrication and Experimental Methods. Microscale chipswere fabricated as follows: A pattern using a computer assisted design(CAD) software (Cadence) was designed and a chrome photomask using aGCA/Mann 3600F Optical Pattern Generator was created. Thishigh-resolution pattern was then transferred to a silicon wafer (3 inchdiameter) containing a thin coat (˜1 μm) of positive photoresist(Shipley 1813) by exposing the wafer to UV light through the photomaskusing a Karl Suss MA6 Contact Aligner. Following exposure, thephotoresist was developed, thus exposing the silicon through thephotoresist layer in the defined pattern. The exposed silicon was etchedto a specified depth (20 to 100 μm) using a PlasmaTherm SLR 770 ICP DeepSilicon Etch System. The photoresist was stripped from the wafer withacetone. Individual 22 mm square microscale chips were diced from thewafer, washed in Nanostrip (Cyantek), rinsed in distilled water, anddried in a drying oven at 170° C.

The surface of the silicon in the organ compartments was treated withcollagen to facilitate cell attachment. Approximately 10 μl of a 1 mg/mlsolution of collagen Type I was deposited onto the surface of themicroscale chip and incubated at room temperature for 30 minutes. Thecollagen solution was removed and the organ compartments were rinsedwith cell culture medium. Cells were dissociated from the tissue culturedishes, cell number was determined, and the concentration was adjustedsuch that there would be a confluent monolayer of cells in each cellcompartment. For example, for the microscale chip described in FIG. 2(hereinabove), 10 μl of a 2,400 cells/μl suspension of the L2 cells wasdeposited onto the lung chamber of the cell chip and 15 μl of a 3,400cells/μl suspension of the H4IIE cells was deposited onto the liverchamber. Cells were allowed to attach in a CO₂ incubator overnight. Oncethe cells were attached, the chip was assembled in acrylic chiphousings. The top of the housings contain fluid interconnects to providecell culture medium to the chip. Stainless steel tubes are connected tomicro-bore pump tubing and inserted into a small hole in the top of amicro-centrifuge tube containing culture medium with or without testcompound. The pump tubing is connected to the peristaltic pump, primedwith this solution, and connected to the inlet ports of the chiphousing. A small section of pump tubing with a stainless steel tubeconnected to the end is connected to the outlet port and the tube isinserted into a small hole in the top of the micro-tube, thus completingthe recirculation fluid circuit. The entire instrument is placed in aCO₂ incubator at 37° C. A schematic diagram of this setup is presentedin FIG. 22.

EXAMPLE 1 Calculations for a System Replicating a Rat

In designing the chip 1000 all necessary chambers were fit onto asilicon chip no larger than 2 cm by 2 cm. This size of chip is easy tomanufacture and is compatible with the sizes of connective tubing andpumping devices intended for use to direct fluid flow. There were alsoseveral other important factors constraining the design of the devicelisted below, along with acceptable values for each variable. This oneembodiment of the device consists of a two compartment system, onecompartment representing the liver of a rat and one compartmentrepresenting the lung of a rat. The total size of the chip is 2 cm by 2cm and consists of an interconnected array of 20 parallel channels 40 μmwide, 10 μm deep and 5 mm long to serve as the “lung” chamber and twoparallel channels 100 μm wide, 20 μm deep and 10 cm long in a serpentineshape to serve as the “liver” chamber. The two organ compartments areconnected by a channel 100 μm wide and 20 μm deep. There are many otherpossible geometries, dimensions, number of chambers, etc. This designwas chosen as one example. TABLE 1 Constraining variables in devicedesign. Constraining variable Acceptable values Chip size 2 cm × 2 cm“Lung” liquid residence time 1.5 seconds “Liver” liquid residence time25 seconds “Other tissues” liquid residence time 204 seconds Number ofeach cell type >10,000 Cell shear stress 8-14 dyne/cm² Channelliquid-to-cell volume ratio 1 to 2Sample CalculationsChannel or Chamber Calculations:These calculations assume we have obtained a flow rate from a previousiteration by the method described above with respect to chip 1000 forsystem 1100.

By this, Q=8.05×10⁵ μm³/trench-second.

The liquid residence time in a trench was then calculated in thefollowing manner: ${\upsilon\quad R} = \frac{V_{Channel}}{Q}$Next, the number of cells in a “cell-length” was calculated$\upsilon_{R} = \frac{\left( {40\quad{µm}} \right) \cdot \left( {10\quad{µm}} \right) \cdot \left( {5000\quad{µm}} \right)}{\left( {8.05 \times 10^{5}\frac{µm}{\sec}} \right)}$υ_(R) = 2.48  sec $N_{Length} = {\frac{Channel\_ Width}{Cell\_ Diameter} + \frac{2 \cdot {Wall\_ Height}}{Cell\_ Diameter}}$$N_{Length} = {\frac{40\quad{µm}}{7.41\quad{µm}} + \frac{20\quad{µm}}{7.41\quad{µm}}}$N _(Length)=7 Cells (Each term is separately rounded down)Then, a channel/chamber cell-length volume was calculated,V _(TCL)=(Cell Diameter)·(Trench Cross Sectional Area)V _(TCL)=(7.4 μm)·(400 μm ²)V_(TCL)=2960 μm³

The cell-length volume was also determined.$V_{CCL} = \frac{\left( N_{Length} \right) \cdot \left( V_{Cell} \right)}{2}$$V_{CCL} = {\left( {7\quad{Cells}} \right) \cdot \left( \frac{320\quad{µm}^{3}}{2\quad{cell}} \right)}$V_(CCL) = 1120  µm³

The liquid cell-length volume is simply the cell cell-length volumesubtracted from the channel/chamber cell-length volume. The ratio of thecell cell-length volume and the liquid cell-length volume gives theliquid-to-cell volume ratio for the system:${{Liquid} - {to} - {{cell}\quad{ratio}}} = \left( \frac{V_{LCL}}{V_{CCL}} \right)$${Ratio} = \left( \frac{{2960\quad{µm}^{3}} - {1120\quad{µm}^{3}}}{1120\quad{µm}^{3}} \right)$Ratio = 1.65The shear forces on individual cells associated with a given flow ratewere determined. Based on the liquid cell-length volume and celldiameter, an average surface area available for liquid to flow throughwas calculated.${{Average}\quad{Liquid}\quad{Surface}\quad{Area}} = \frac{V_{LCL}}{D_{Cell}}$$A_{LS} = \frac{\left( {1844\quad{µm}^{3}} \right)}{7.41\quad{µm}}$A_(LS) = 249  µm²An average linear velocity of fluid in the channel was then calculated.$V_{avg} = \frac{Q}{A_{LS}}$$V_{avg} = \frac{\left( {8.05 \times 10^{5}\frac{{µm}^{3}}{\sec}} \right)}{249\quad{µm}^{2}}$$V_{avg} = {3.23 \times 10^{3}\frac{µm}{\sec}}$Assuming laminar flow, Stokes' law was used for calculating the drag ona sphere to estimate the total shear force experienced by an individualcell,$\Gamma_{s} = \frac{\left( {3{\pi\eta}\quad D_{Cell}V_{avg}} \right)}{A_{Cell}}$$\Gamma_{s} = \frac{\begin{pmatrix}{3 \cdot \pi \cdot \left( {9.60 \times 10^{- 4}\frac{N - \sec}{m^{2}}} \right) \cdot} \\{\left( {7.41\quad{µm}} \right) \cdot \left( {3.23 \times 10^{3}\frac{µm}{\sec}} \right)}\end{pmatrix}}{\frac{4}{2} \cdot \pi \cdot \left( \frac{7.41\quad{µm}}{2} \right)^{2}}$$\Gamma_{s} = {12.6\frac{dyne}{{cm}^{2}}}$Next, the actual residence time of the liquid in a channel/chamber wasverified and calculated to total number of cells in the channel/chamber,$N_{Cells} = \frac{L_{Trench} \cdot N_{Trenches} \cdot N_{Length}}{D_{Cell}}$$N_{Cells} = \frac{\left( {5000\quad{µm}} \right) \cdot \left( {20\quad{trenches}} \right) \cdot \left( {7\quad{Cells}} \right)}{\left( {7.41\quad{µm}} \right)}$N_(Cells) = 9.45 × 10⁴  CellsI.B. Membrane Oxygenation Calculations:

The area of silicone membrane for oxygenation was determined in thefollowing manner:

First, approximate the Oxygen Uptake Rate (OUR) for the cells:OUR = q_(O₂) ⋅ X${OUR} = {\left( {7.00\frac{{µgO}_{2}}{{10^{6}\quad{cells}} - {hr}}} \right) \cdot \left( {2 \times 10^{5}\quad{cells}} \right)}$${OUR} = {4.4 \times 10^{- 5}\frac{m\quad{mol}\quad O_{2}}{hr}}$Then calculate the partial pressure of oxygen on the inside of themembrane to determine if it is sufficient to re-oxygenate the liquidmedium. This was done using an equation for the flux of a gas through aporous membrane, where Q is the membrane permeability. J represents theflux of gas into the cells, and z is the thickness of the membrane:${J_{O_{2}}A_{Membrane}} = {{OUR} = \frac{Q_{O_{2}} \cdot \left( {P_{O_{2},{Out}} - P_{O_{2},{In}}} \right)}{z}}$${\left( {4.4 \times 10^{- 5}\frac{m\quad{mol}\quad O_{2}}{hr}} \right) \cdot \left( {55\quad{mm}^{2}} \right)} = \frac{\begin{matrix}{\left( {5.00 \times 10^{- g}\frac{\left\lbrack {{{cm}^{3}({STP})} \cdot {cm}} \right\rbrack}{\left( {{{cm}^{2} \cdot s \cdot {cm}}\quad{Hg}} \right)}} \right) \cdot} \\\left( {P_{O_{2}{Out}} - {16\quad{cm}\quad{Hg}}} \right)\end{matrix}}{0.05\quad{cm}}$ P_(O₂, Out) = 15.5  cm  HgThis pressure is sufficient to saturate the liquid medium with oxygen inthe 200 seconds it is in contact with the membrane. The area of membranewas determined in an iterative manner so as to maximize the insideoxygen partial pressure.Principle Design Calculations

Rat Model: Lung (L2) Liver (H4IIE) Primary cell characteristics Surfacearea (cm²/organ) 4890 21100  Cell volume (μm³/cell) 320 4940  Platingarea (μm²/cell) 320 988 Cell Diameter (μm) 7.41   18.5Stokes' law: 3πηDU = F_(D) (Plating area is the inverse ofexperimentally determined saturation densities for L2 and H4IIE cells.)

LUNG CELL CALCULATIONS: Calculation of cell and liquid volumes in onecell-length of channel/chamber: Cell diameter 7.41 μM (a cell-lengthCell volume 320 μm³/cell included the diameter Channel width 40 μm ofthe cell as well as Channel depth 10 μm spacing on either side Spacingbetween channels 30 μm equal to the “distance Channel X-sectional area400 μm² between cells”) Cells across channel 5 Cells on side of channel1 Total cells in one 7 cell-length Channel cell-length volume 2964 μm³Cell cell-length volume 1120 μm³ Liquid cell-length volume 1844 μm³Liquid-to-cell volume ratio 1.65 Determination of liquid velocity andshear on individuals cells: Viscosity of cell plasma 9.60E−04 N-s/m²medium Number of channels 20 (this number picked to give adequate # ofcells and feasible flows) Liquid flow rate per 8.05E+05 μm³/sec (thisnumber channel picked to give a stress of 12 dyne) Average liquidsurface area 249 μm² Average liquid linear 3.23E+03 μM/SEC Velocity, U3.23E−03 M/SEC Drag force on individual 1.08E−10 Newtons (for a half-cell 1.08E−04 μN sphere) 1.08E−05 dyne Surface area of individual8.63E+01 μm² (for a half- cell 8.63E−07 cm² sphere) Shear stress onindividual 12.6 dyne/cm² (This result cell assumes smooth half-spherical geometry for the cells; it is likely the actual number issmall due to larger surface area or surface irregularities) Total flowrate 1.61E+07 μm³/sec Desired residence time 1.5 seconds Channel length5 mm (this number is chosen to give the desired residence time) TotalChannel liquid 2.49E+07 μm³ volume Actual Residence time 1.55 secondsTotal number of cells 9.45 + 04 cells

LIVER CELL CALCULATIONS: Calculation of cell and liquid volumes in onecell-length of channel/chamber Cell diameter 18.5 μm Cell volume 4940μm³/cell Channel width 100 μm Channel depth 20 μm Spacing betweenchannels 50 μm Channel X-sectional area 2000 μm² Cells across channel 5Cells on side of channel 1 Total cells in one cell-length 7 Channelcell-length volume 36918 μm³ Cell cell-length volume 17290 μm³ Liquidcell-length volume 19628 μm³ Liquid-to-cell volume ratio 1.14Determination of liquid velocity and shear on individual cells:Viscosity of cell plasma 9.60E−04 N-s/m² medium Total liquid flow ratefrom 1.61E+07 μm³/sec (from above Lung Calcs. calcs.) Number of channels2 Liquid flow rate per channel 8.05E+06 μm³/sec Average liquid surfacearea 1063 μm² Average liquid linear U7.57E+03 μm/sec velocity 7.57E−03m/sec Drag force on individual cell 6.32E−10 Newtons Stokes' law:6.32E−05 dyne 3πηDU = F_(D) Surface area of individual 535.24 μm² cell5.35E−06 cm³ Shear stress on individual 11.81 dyne/cm² cell Desiredresidence time 25 sec channel length 100 mm Total Channel liquid volume4.00E+08 μm³ Actual Residence time 24.86 sec Total number of cells7.58E+04 cells

Lung 1.5 sec Liver 25 sec Other Tissues 204 sec

Volume Blood Flow Rate (mL/min) (mL) Lung 73.3 1.2 Liver 18.3 7.4 OtherTissues 55 190 Preliminary flow rate 0.85 μL/min 0.0142 μL/sec

1 μm 1 μL 0.000001 m 1.00E−06 L 1.00E−09 m³ 1.00E+09 μm³

Preliminary Residence Time Calculations for Liver/Lung: Channel Depth310 μm Channel Width 500 μm Channel X-sectional Area 0.155 mm² 155000μm² Cells per area 3200 cells/mm² Channel Surface Residence VolumeChannel Area Max # Time (sec) (μL) Length (mm) (mm²) cells Lung 1.50.02125 0.1 6.85E+01 2.58E+04 Liver 25 0.4 2   1.14E+03 3.66E+06Preliminary Residence Time Calculations for Other Tissues: Channel Depth50 μm Channel Width 2000 μm Channel X-sectional Area 0.1 mm² 100000 μm²Residence CHANNEL VOLUME Channel Length Surface Area Time (sec) (μL)(mm) (mm²) 204 2.89 29 57.8

EXAMPLE 2 A Four Organ Compartment Chip

A chip was designed to consist of four organ compartments—a “liver”compartment to represent an organ responsible for xenobiotic metabolism,a “lung” compartment representing a target tissue, a “fat” compartmentto provide a site for bio-accumulation of hydrophobic compounds, and an“other tissues” compartment to assist in mimicking the circulatorypattern in non-metabolizing, non-accumulating tissues (FIG. 15). Theseand other organ compartments (e.g., kidney, cardiac, colon or muscle)can be fully modularized as CAD files and can be fabricated in anyconfiguration or combination. The device itself can be produced in anynumber of substrates (e.g., silicon, glass, or plastic).

Once the cells were seeded in the appropriate compartments, the chip wasassembled in a Lucite manifold. This manifold holds four chips andcontained a transparent top so the cells could be observed in situ. Thetop contained fluid interconnects to provide cell culture medium to thechip. The culture medium was pumped through the chip using a peristalticpump at a flow rate of 0.5 μl/min. Culture medium was re-circulated in aclosed loop consisting of a fluidic reservoir (˜15 to 50 μl totalvolume), micro-bore tubing, and the compartments and channels of thechip.

Using a three compartment system with human HepG2-C3A cells in the livercompartment and HT29 colon cancer cells in the target tissuescompartment, it was found that cells remain viable under continuousoperation for greater than 144 hours. HepG2-C3A cells are a wellcharacterized human liver cell line known to express various livermetabolizing enzymes at levels comparable to fresh primary humanhepatocytes. In these experiments, cells were seeded in the appropriatecompartments and a specially formulated cell culture medium wasre-circulated through the system for up to 144 hours. At various timepoints, the culture medium was switched to PBS containing LIVE/DEADfluorescent reagent (a dual fluorescent stain, [Molecular Probes, Inc.,Eugene, Oreg., USA]) for 30 minutes. Cells were visualized under afluorescent microscope and fluorescent images of identical fields wereobtained using the appropriate filter sets. Living cells fluorescedgreen whereas dead cells were red (data not shown).

EXAMPLE 3 Drug Metabolism in the Chip

The metabolism of two widely used prodrugs, tegafur and sulindacsulfoxide, was studied using a microscale chip comprising threecompartments, liver, target tissue, and other tissues. Both prodrugsrequire conversion to an active metabolite by enzymes present in theliver, and have a cytotoxic effect on a target organ. For the prodrugsulindac sulfoxide, its anti-inflammatory and cancer chemopreventiveproperties are derived from its sulfide and sulfone metabolites,catalyzed by the liver enzyme sulfoxide reductase. The sulfidemetabolite (and a second sulfone metabolite) have been demonstrated toinduce apoptosis in certain cancer cells (e.g., colon cancer).

A proper treatment regimen requires administration of its prodrug,tegafur [5-fluoro-1-(2-tetrahydrofuryl)-2,4(1H,3H)-pyrimidi-nedione] as5-FU itself is quite toxic to normal cells. Unlike sulindac however,tegafur is converted to 5-FU in the liver primarily by cytochrome P4502A6.

To test the efficacy of sulindac, the microscale chip was seeded withHepG2-C3A cells in the liver compartment and HT29 human colon cancercells in the target tissue compartment. One hundred micromoles ofSulindac (need manufacturer) was added to the re-circulating medium for24 hours and the chip was treated as described above—living cellsfluoresced green and dead cells fluoresced red (data not shown). In theabsence of the HepG2-C3A liver cells, minimal levels of cell death(similar to vehicle control) was observed. These results demonstratethat a drug can be metabolized in the liver compartment and consequentlycirculate to a target where its metabolite(s) induce a biological effectmuch as it would in a living animal or human.

The cancer therapeutic pro-drug tegafur was tested in the microscalechip system. For efficacy, tegafur requires metabolic activation bycytochrome P450 enzymes present in the liver to its active form,5-fluorouracil (5-FU) (Ikeda, K., Yoshisue, K., Matsushima, E.,Nagayama, S., Kobayashi, K, Tyson, C. A., Chiba, K., and Kawaguchi, Y.(2000). Bioactivation of tegafur to 5-fluorouracil is catalyzed bycytochrome P-450 2A6 in human liver microsomes in vitro. Clin. CancerRes., 6, 4409-4415; Komatsu, T., Yamazaki, H., Shimada, N., Nakajima,M., and Yokoi, T. (2000). Roles of cytochromes P450 1A2, 2A6, and 2C8 in5-fluorouracil formation from tegafur, an anticancer prodrug, in humanliver microsomes. Drug Met. Disp., 28, 1457-1463; Yamazaki, H., Komatsu,T., Takemoto, K., Shimada, N., Nakajima, M., and Yokoi, T. (2001). Ratcytochrome P450 1A and 3A enzymes involved in bioactivation of tegafurto 5-fluorouracil and autoinduced by tegafur liver microsomes. Drug Met.Disp., 29, 794-797. A proper therapeutic regimen requires administrationof its pro-drug, tegafur, as 5-FU itself is very toxic to normal cells.5-FU is currently the most effective adjuvant therapy for patients withcolon cancer (Hwang, P. M., Bunz, F., Yu, J., Rago, C., Chan, T. A.,Murphy, M. P., Kelso, G. F., Smith, R. A. J., Kinzler, K. W., andVogelstein, B. (2001). Ferredoxin reductase affects p53-dependent,5-fluorouracil-induced apoptosis in colorectal cancer cells. Nat. Med.,7, 1111-1117.) Like most chemotherapeutic agents, 5-FU induces markedapoptosis in sensitive cells through generation of reactive oxygenspecies (Hwang, P. M., Bunz, F., Yu, J., Rago, C., Chan, T. A., Murphy,M. P., Kelso, G. F., Smith, R. A. J., KinzIer, K. W., and Vogelstein, B.(2001). Ferredoxin reductase affects p53-dependent,5-fluorouracil-induced in colorectal cancer cells. Nat. Med., 7,1111-1117).

To measure the cytotoxic effects of tegafur against colon cancer cells,the microscale chip was prepared with HepG2-C3A cells in the livercompartment and HCT-116 human colon cancer cells in the target tissuecompartment. Tegafur was added to the recirculating medium at variousconcentrations for 24 hours and the cells labeled with Hoechst 33342, amembrane permeable DNA dye, and ethidium homodimer, a membraneimpermeable DNA dye (see Methods Section). All cells fluoresce blue, butdead cells were marked by the fluorescent red ethidium homodimer (datanot shown). Tegafur was cytotoxic to HCT-116 cells in a dose-dependentfashion in this microscale chip system, while it was ineffective withthe traditional cell culture assay (FIGS. 16A and 16B). In addition,while 5-FU triggered cell death in the traditional cell culture assay,cytotoxicity was not observed until after 48 hours of exposure comparedto 24 hours of exposure to tegafur with the microscale chip.

To demonstrate that the liver compartment was responsible for thebio-activation of tegafur, the microscale chips were seeded with HCT-116cells only. No cells were in the liver compartment. Tegafur or 5-FU wasadded to the re-circulating culture medium for 24 hours and the chip wastreated as described above (data not shown). Tegafur did not causesignificant cell death of the HCT-116 cells in the absence of a livercompartment while the active metabolite 5-FU caused substantial celldeath. Further, when HT-29 colon cancer cells are substituted forHCT-116, tegafur was ineffective (data not shown). This was likely dueto the mutant p53 present in HT-29 cells, which is necessary for 5-Fcytotoxicity. Together, these experiments demonstrate that tegafur, likesulindac, was metabolized to an active drug in the liver compartmentwhere it circulated to another organ compartment to eliminate the cancercells. These effects were mechanistically distinguishable with thechip—sulindac was effective even in the absence of an active p53,whereas tegafur was not.

EXAMPLE 4 Multiple Cell Cultures in a Single Organ Compartment

It is also possible to use a mixture of multiple cell types in a singleorgan compartment. In one study, the hepatocyte cell line HepG2/C3A(from ATCC) is used in the liver compartment. The cells are propagatedin McCoy's 5A medium with 1.5 mM L-glutamine 1.5 g/L sodium bicarbonateand 10% fetal bovine serum. To more closely mimic an in vivo organ, amixture of primary hepatocytes and fibroblasts can be used at a 1 to 2ratio along with macrophages (Kupffer cells).

In another example, a mixture of cells or cell lines derived from lungepithelial cells is used to more closely mimic the lung tissue. Thisincludes a mixture of type I epithelial cells, type II epithelial cells(granular pneumocytes), fibroblasts, macrophages and mast cells.

EXAMPLE 5 Optimization of Tissue Culture Conditions in the Chip-BasedSystem

A tissue culture medium compatible with two different rat cell culturelines, H4IIE (a rat liver cell line) and L2 (a rat lung cell line) wasdeveloped. Preliminary experiments indicated that a 1:1 mixture of DMEMand Hams F12K medium supplemented with 2 mM L-glutamine, 1 mM sodiumpyruvate and 10% fetal bovine serum (FBS) maintained the viability ofboth H4IIE cells and L2 cells for up to 20 hours of continuous operationin a microscale chip. This media formulation was used for all rat-basedmicroscale chip studies.

The proper human liver cell line that realistically mimics human liverfunction was selected. Additionally, the optimum cell culture mediumformulation for maintaining human cell lines on a microscale chip wasdetermined. The basal expression levels of three key cytochrome P450(CYP) isoforms (1A2, 3A4, and 2D6) in HepG2 and HepG2/C3A (a HepG2subclone) cell lines were examined. CYP-1A2, 2D6, and 3A4 were examinedbecause they account for the metabolism of 80-90% of all known drugs(Hodgson, J., (2001). ADMET—turning chemicals into drugs. Nat. Biotech.,19, 722-726. The C3A subclone of the HepG2 liver cell line was examinedas this cell line has been reported to be a highly selected cell lineexhibiting more “liver-like” characteristics, particularly much higherCYP expression compared to the parental cell line (Kelly, J. H. (1994).Permanent human hepatocyte cell line and its use in a liver assistdevice (LAD). U.S. Pat. No. 5,290,684). The RT-PCR analysis confirmedthat basal CYP levels in HepG2/C3A cells were significantly greater thanHepG2 parentals and comparable to adult human liver (FIG. 23).

HepG2/C3A cells were used as a liver surrogate in all subsequentexperiments. To select a common media for use during microscale chipexperiments, the components of a number of media were compared (DMEM,McCoy's 5a, RPMI 1640, MEM, F12, F12K, Waymouth's, CMRL, MEM, andIscove's modified Dulbecco's medium). Analysis of the inorganic salt,glucose, amino acid composition, and vitamin content suggested thatEMEM, DMEM, McCoy's 5a and RPMI were the most suitable “common” media ofthe media examined. After several passages, cells were then split andsub-cultured in the following media:

Eagle's Minimum Essential medium (EMEM) with Earle's balanced saltssolution, 2 mM L-glutamine, 1.0 mM sodium pyruvate, 0.1 mM nonessentialamino aids, 1.5 g/L sodium bicarbonate, and 10% fetal bovine serum.

Dulbecco's modified Eagle's medium (DMEM) with 4 mM L-glutamine, 4.5 g/Lglucose, 1.5 g/L sodium bicarbonate, and 10% fetal bovine serum.

McCoy's 5a medium (McCoy's) with 1.5 mM L-glutamine 1.5 g/L sodiumbicarbonate and 10% fetal bovine serum.

RPMI 1640 medium (RPMI) with 2 mM L-glutamine, 4.5 g/L glucose, 1.0 mMsodium pyruvate, 1.5 g/L sodium bicarbonate.

Growth curves for both cell lines in each media were then determined asdescribed in the Methods section (FIG. 24). DMEM was found to beinappropriate for the HepG2/C3A cells, as significant changes incellular morphology and adhesion after ˜5 passages were observed (notshown). Similarly, a significant decrease in HepG2/C3A and HCT116viability and growth after 3 days in RPMI was noticed. Both cell linesgrew well in McCoy's and EMEM compared to their preferred medium.

Next, the expression levels of these CYP isoforms in HepG2/C3A cellsgrowing in either EMEM or McCoy's using RT-PCR were investigated (seeMethods section) (FIG. 25). The results indicated that EMEM was superiorto McCoy's for maintaining CYP expression and the preferred media forHepG2/C3A. The effect of different growth substrates on CYP expressionwas studied (FIG. 26). A comparison of silicon treated with eitherpoly-D-lysine or collagen as the attachment substrate against cellsgrown on standard tissue culture treated polystyrene was performed.Together, the results indicated that EMEM supported the growth of bothHepG2/C3A and HCT116 cells and that collagen was the preferred substratebased on RT-PCR CYP expression analysis.

Using these conditions, the long term cell viability of these cells,HepG2/C3A and HCT116, was studied under continuous operation in themicroscale chip system. Using a three compartment system with humanHepG2/C3A cells in the liver compartment and HCT116 colon cancer cellsin the target tissues compartment, it was demonstrated that cells remainviable under continuous operation for greater than 144 hours. In theseexperiments, cells were seeded in the appropriate compartments and EMEMwas re-circulated through the system for up to 144 hours. At varioustime points (6, 24, 48, 72, 96, 120 and 144 hr), total live or deadcells were visualized using LIVE/DEAD stain (data not shown). Cells werevisualized under a fluorescent microscope and fluorescent images ofidentical fields were obtained using the appropriate filter sets. Livingcells fluoresced green whereas dead cells were red (data not shown).

EXAMPLE 6 Assay for Detection of Cytotoxicity on a Microscale Chip

Trypan blue is the most common stain used to distinguish viable cellsfrom nonviable cells; only nonviable cells absorb the dye and appearblue. Conversely, live, healthy cells appear round and refractilewithout absorbing the blue dye. Experiments were performed using trypanblue to determine cell viability in a microscale chip. Although trypanblue (see Methods section) is easy to use and requires only a lightmicroscope to visualize, viable cells will absorb trypan blue over time,which can affect results. In addition, trypan blue has a higher affinityfor serum proteins than for cellular proteins, thus the background isdark when using serum-containing media. Therefore, alternative methodsto distinguish viable cells from dead cells were studied.

The LIVE/DEAD assay was optimized (see Methods section) using cellsgrown on glass coverslips. Briefly, HepG2/C3A cells were seeded ontopoly-D-lysine treated glass coverslips and treated with and without 1 μMstaurosporine for 24 hours. Staurosporine is a broad-spectrum proteinkinase inhibitor and is known to induce apoptosis in a variety of celltypes (Smyth, P. G., Berman, S. A., and Bursztajn, S. (2002). Markers ofapoptosis: methods for elucidating the mechanism of apoptotic cell deathfrom the nervous system. Biotechniques, 32, 648-665). Coverslips werewashed with phosphate buffered saline (PBS) and LIVE/DEAD reagents wereadded and incubated at room temperature for 30 minutes. The coverslipswere removed and visualized (data not shown). Staurosporine was found toclearly cause cell death of HepG2/C3A cells (data not shown).

The assay for detection of cytotoxicity on the microscale chip systemwas then optimized. Microscale chip cell chips were seeded withHepG2/C3A cells in the liver compartment and HCT116 cells in the targettissues compartment as described in the Methods section. Cell chips wereloaded onto the microscale chip system and treated with and without 1 μMstaurosporine as described above. After a 24-hour incubation, therecirculating medium was switched to PBS, allowed to flow through thesystem to waste for 30 minutes, then switched to PBS containing theLIVE/DEAD reagents and flowed through the system for an additional 30minutes. The acrylic housing containing the cell chips was removed fromthe system and placed under a stereofluorescence microscope and the cellchip was visualized through the transparent top of the housing (data notshown). Cells were visualized under a fluorescent microscope andfluorescent images of identical fields were obtained using theappropriate filter sets. Living cells fluoresced green whereas deadcells were red (data not shown). Significant cell death of the HCT116cells was caused by 1 μM staurosporine after a 24 hour treatmentcompared to untreated control cell chips (data not shown).

EXAMPLE 7 Chip-Based Assays to Detect the Occurrence of Cell Death andDistinguish Between Apoptosis or Necrosis

Two different assays to detect apoptosis were investigated. The firstassay was the immunofluorescence-based terminal deoxynucleotidyltransferase BrdU nick end labeling (TUNEL) technique available in kitform as APOPTAG™ (Intergen Co., Massachusetts) (see Methods section).The assay was first optimized using cells grown on glass coverslips.Briefly, HepG2/C3A cells were seeded onto poly-D-lysine treated glasscoverslips and treated with and without staurosporine. Coverslips wereprocessed as described (see Methods section). Various staurosporineconcentrations and treatment times were tested, and the resultsindicated that 1 μM staurosporine caused significant apoptosis comparedto untreated controls after a 24-hour incubation (data not shown). Next,the assay for detection of apoptosis on the microscale chip system wasoptimized and a comparison of the APOPTAG™ method to the LIVE/DEADstaining technique was performed. The microscale cell chips were seededwith HepG2/C3A cells in the liver compartment and HCT116 cells in thetarget tissues compartment as described in the Methods section. Cellchips were loaded onto the microscale chip system and treated with andwithout 1 82 M staurosporine as described above. After a 24-hourincubation, the recirculating medium was switched to PBS for 30 minutes.Half the cell chips were removed from the housing and the APOPTAG™ assaywas performed as described above. The other cell chips were left in themicroscale chip system and subjected to the LIVE/DEAD staining techniqueas previously described. Cells were visualized under a fluorescentmicroscope and fluorescent images of identical fields were obtainedusing the appropriate filter sets. Living cells fluoresced green whereasdead cells were red (data not shown). Both techniques produced verysimilar results, ie., a 24 hour exposure to 1 μM staurosporine inducedsignificant apoptosis (or cytotoxicity) to the HCT116 cells compared tountreated controls (data not shown).

The annexin V-FITC was used to detect apoptosis in the microscale chipsystem as described in the Methods section. Briefly, the microscale chipcell chips were seeded with HepG2/C3A cells in the liver compartment andHCT116 cells in the target tissues compartment. Cell chips were loadedonto the microscale chip system and treated with and without 1 μMstaurosporine as described above. After a 6-hour incubation, therecirculating medium was switched to PBS containing Annexin V-FITC andHoechst 33342 and allowed to flow through the system for 30 minutes.Cell chips were removed from the acrylic housing and visualized under afluorescent microscope. Cells were visualized under a fluorescentmicroscope and fluorescent images of identical fields were obtainedusing the appropriate filter sets. Living cells fluoresced green whereasdead cells were red (data not shown). 1 μM staurosporine causedsignificant apoptosis after a 6-hour treatment compared to untreatedcontrol cell chips (data not shown).

EXAMPLE 8 Use of Naphthalene as a Model Toxicant

Naphthalene was used to study toxicology because enzymatic conversion inthe liver is required for lung toxicity. Therefore, the effects ofnaphthalene on a rat lung cell line were studied. These experiments useda three compartment (liver, lung, and other tissues) rat-basedmicroscale chip with H4IIE cells in the liver compartment and rat L2cells in the lung compartment. Microscale chips were fabricated andprepared for experiments as described in the Method section.

The microscale chip system was operated for 20 hours in the presence orabsence of 250 μg/ml naphthalene before switching to PBS containingtrypan blue. This solution was re-circulated through the cell chip for30 minutes and the chip visualized under a light microscope (see Methodssection). Naphthalene caused significant cell death of the rat L2 cellsin the lung compartment of the cell chip while no cell death wasobserved in the absence of naphthalene (data not shown). No cell deathwas observed in the H4IIE cell compartment with or without naphthaleneor in the L2 cell compartment in the absence of H4IIE cells (data notshown).

These results demonstrate that naphthalene is activated in the “liver”compartment and the toxic metabolites circulate to the “lung” and causecell death. These results are consistent with data obtained with thebenchtop CCA device and expected from the PBPK model (Sweeney, L. M.,Shuler, M. L., Babish, J. G., and Ghanem, A. (1995). A cell cultureanalogue of rodent physiology: application of napthalene toxicology.Toxicol. in Vitro, 9, 307-316).

EXAMPLE 9 A Human Microscale Chip Prototype

A human biochip prototype was prepared that contained compartments forlung, target tissues, and other tissues. The dimensions of thecompartments and channels were as follows:

Inlet: 1 mm by 1 mm

Liver: 3.2 mm wide by 4 mm long

Target Tissues: 2 mm by 2 mm

Other Tissues: 340 μm wide by 110 mm long

Outlet: 1 mm by 1 mm

Channel Connecting Liver to Y connection: 440 μm wide

Channel from Y connection to Target Tissue: 100 μm wide

The human biochip prototype is fabricated as described previously. Theplacement of the organ compartments is intended to simulate exposure toa compound (drug) that has been ingested orally. When a compound isorally ingested it is absorbed into the blood from the small or largeintestine. From here it circulates directly to the liver via the hepaticportal vein then gets distributed throughout the body (FIG. 27).Therefore, with this design, the liver is the first organ compartment,followed by a split to other tissues a compartment and a chamber for thetarget tissue. The other tissues compartment represented distributionand hold-up of blood in the body, the target tissue compartmentrepresents the therapeutic target of interest (e.g., colon cancer cellsrepresenting a colon tumor.

Conclusion

The invention provides a pharmacokinetic-based culture device andsystems, usually including a first cell culture chamber having areceiving end and an exit end, and a second cell culture chamber havinga receiving end and an exit end, and a conduit connecting the exit endof the first cell culture chamber to the receiving end of the secondcell culture chamber. Preferably the device is chip-based, ie., it ismicroscale in size. A culture medium can be circulated through the firstcell culture chamber, through the conduit and through the second culturechamber. The culture medium may also be oxygenated at one or more pointsin the recirculation loop.

The device may include a mechanism for communicating signals fromportions of the device to a position off the chip, e.g., with awaveguide to communicate signals from portions of the device to aposition off the chip. Multiple waveguides can be present, e.g., a firstwaveguide communicating signals from the first chamber, and a secondwaveguide communicating signals from a second chamber, and so forth.

In one embodiment, at least one of the first cell culture chamber andthe second cell culture chamber is three dimensional. In anotherembodiment, both the first cell culture chamber and the second cellculture chamber are three dimensional.

The device for maintaining cells in a viable state also includes a fluidcirculation mechanism, may be a flow through fluid circulation mechanismor a fluid circulation mechanism that recirculates the fluid. The devicefor maintaining cells in a viable state also includes a fluid path thatconnects at least the first compartment and the second compartment. Inan embodiment, a debubbler removes bubbles in the flow path. The devicecan further include a pumping mechanism. The pumping mechanism may belocated on the substrate.

A method is provided for sizing a substrate to maintain at least twotypes of cells in a viable state in at least two cell chambers. Themethod includes the steps of determining the type of cells to be held onthe substrate, and applying the constraints from a physiologically basedpharmacokinetic model to determine the physical characteristics of thesubstrate. The step of applying the constraints from a physiologicallybased pharmacokinetic model includes determining the type of chamber tobe formed on the substrate, which may also include determining thegeometry of at least one of the cell chambers and determining thegeometry of at a flow path interconnecting two cell chambers. The stepof applying the constraints from a physiologically based pharmacokineticmodel may also include determining the flow media composition of theflow path.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

It is to be understood that the above description is intended to beillustrative, and not restrictive. Many other embodiments will beapparent to those of skill in the art upon reviewing the abovedescription. The scope of the invention should, therefore, be determinedwith reference to the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1. A pharmacokinetic-based microscale culture device, comprising: afirst microscale chamber containing a first type of cell underconditions where the first type of cell provides at least onepharmacokinetic parameter value comparable to a value obtained for thesame type of cell in vivo, wherein the first chamber comprises a firstinlet and a first outlet for flow of culture medium; a second microscalechamber containing a second type of cell under conditions where thesecond type of cell provides at least one pharmacokinetic parametervalue comparable to a value obtained for the same type of cell in vivo,wherein the second chamber comprises a second inlet and a second outletfor flow of culture medium; and a microfluidic channel interconnectingthe first and second chambers.
 2. The culture device of claim 1, furthercomprising one or more additional microscale chambers containing anadditional type of cell under conditions where the additional cellprovides at least one pharmacokinetic parameter value comparable to avalue obtained for the same type of cell in vivo, wherein the one ormore additional chambers comprise an inlet and outlet for flow ofculture medium.
 3. The culture device of claim 1, further comprisingculture medium.
 4. The culture device of claim 3, wherein the culturemedium flows through the chambers.
 5. The culture device of claim 3,wherein the culture medium flows re-circulated through the chambers. 6.The culture device of claim 3, further comprising a pumping mechanism.7. The culture device of claim 6, wherein the pumping mechanism isintegrated in the device.
 8. The culture device of claim 6, wherein thepumping mechanism is external to the device.
 9. The culture device ofclaim 1, further comprising a debubbler located within the microfluidicchannel.
 10. The culture device of claim 1, further comprising adebubbler located externally to the device.
 11. The culture device ofclaim 1, wherein at least one of the pharmacokinetic parameters is ameasurement of interaction between cells, liquid residence time, liquidto cell ratio, metabolism by cells, or shear stress.
 12. The culturedevice of claim 1, further comprising at least one sensor for obtainingsignals from cultured cells.
 13. The culture device of claim 12, whereinthe at least one sensor is a biosensor.
 14. The culture device of claim12, wherein the at least one sensor comprises a waveguide.
 15. Theculture device of claim 1, wherein the device is microfabricated. 16.The culture device of claim 1, wherein the device is manufactured from amicrofabricated master.
 17. The culture device of claim 1, wherein atleast one of the chambers provides for three-dimensional growth ofcells.
 18. The culture device of claim 1, wherein at least one of thechambers contains a plurality of cells.
 19. The culture device of claim1, wherein at least one of the chambers contains a tissue biopsy. 20.The culture device of claim 1, wherein at least one of the chamberscontains a cross-section of a tissue.
 21. The culture device of claim 19or 20, wherein the tissue is healthy or diseased.
 22. The culture deviceof claim 19 or 20, wherein the tissue is an artery, vein,gastrointestinal tract, esophagus, or colon.
 23. The culture device ofclaim 1, wherein at least one of the chambers contains a cross-sectionof an organ.
 24. The culture device of claim 23, wherein the organ ishealthy or diseased.
 25. The culture device of claim 23, wherein theorgan is a heart, brain, kidney, lung, or muscle.
 26. The culture deviceof claim 1, wherein at least one of the chambers contains circulating oradherent cells.
 27. The culture device of claim 1, wherein at least oneof the chambers contains eukaryotic cells.
 28. The culture device ofclaim 27, wherein the eukaryotic cells are plant or animal cells. 29.The culture device of claim 28, wherein the cells are mammalian cells.30. The culture device of claim 1, wherein at least one of the chamberscontains prokaryotic cells.
 31. The culture device of claim 1, whereinthe cells are primary cells.
 32. The culture device of claim 1, whereinthe cells are tumor cells.
 33. The culture device of claim 1, whereinthe cells are stem cells.
 34. The culture device of claim 1, wherein thecells are genetically altered, transformed or immortalized cells.
 35. Apharmacokinetic-based culture system of cells grown in a microscaleculture device, comprising: a first chamber comprising a first cell typemaintained under conditions providing at least one pharmacokineticparameter value comparable to values obtained for the cells in vivo; asecond chamber of different geometry than the first chamber comprising asecond cell type maintained under conditions providing at least onepharmacokinetic parameter value comparable to values obtained for thecells in vivo; wherein the first and second chambers are interconnectedby fluidic channels; and an inlet and outlet for recirculation ofculture medium.
 36. The culture system of claim 35, comprising multipleinterconnected devices.
 37. The culture system of claim 35, wherein theat least one pharmacokinetic parameter a measurement of interactionsbetween cells, liquid residence time, liquid to cell ratios, metabolismby cells, and shear stress.
 38. The culture system of claim 35, whereineach of the chambers provides for at least two pharmacokinetic parametervalues comparable to values obtained for a cell of interest in vivo. 39.The culture system of claim 35, further comprising a pumping mechanism.40. The culture system of claim 35, further comprising a debubblerlocated within a microfluidic channel.
 41. The culture system of claim35, further comprising at least one sensor for obtaining signals fromcultured cells.
 42. The culture system of claim 41, wherein the at leastone sensor is a biosensor.
 43. The culture system of claim 41, whereinthe at least one sensor comprises a waveguide.
 44. The culture system ofclaim 35, wherein the device is microfabricated.
 45. The culture systemof claim 35, wherein the device is manufactured from a microfabricatedmaster.
 46. The culture system of claim 35, wherein at least one of thechambers provides for three-dimensional growth of cells.
 47. The culturesystem of claim 35, wherein at least one of the chambers contains aplurality of cells.
 48. The culture system of claim 35, wherein at leastone of the chambers contains a tissue biopsy.
 49. The culture system ofclaim 35, wherein at least one of the chambers contains a cross-sectionof a tissue.
 50. The culture system of claim 48 or 49, wherein thetissue is healthy or diseased.
 51. The culture device of claim 48 or 49,wherein the tissue is an artery, vein, gastrointestinal tract, esophagusor colon.
 52. The culture system of claim 35, wherein at least one ofthe chambers contains a cross-section of an organ.
 53. The culturesystem of claim 52, wherein the organ is healthy or diseased.
 54. Theculture system of claim 48, wherein the organ is a heart, brain, kidneyor lung.
 55. The culture system of claim 35, wherein at least one of thechambers contains circulating or adherent cells.
 56. The culture systemof claim 35, wherein at least one of the chambers contains eukaryoticcells.
 57. The culture system of claim 56, wherein the eukaryotic cellsare plant or animal cells.
 58. The culture system of claim 35, whereinat least one of the chambers contains prokaryotic cells.
 59. The culturesystem of claim 57, wherein the cells are mammalian cells.
 60. Theculture system of claim 35, wherein the cells are primary cells.
 61. Theculture system of claim 35, wherein the cells are tumor cells.
 62. Theculture system of claim 35, wherein the cells are genetically altered,transformed or immortalized cells.
 63. The culture system of claim 35,wherein the cells are stem cells.
 64. A method for determining theeffect of an input variable on a pharmacokinetic-based culture system ofcells, the method comprising: contacting the culture system of claim 35with an input variable; and monitoring at least one output parameter.65. The method of claim 64, wherein the step of monitoring the at leastone output parameter comprises obtaining information from at least onesensor in the device.
 66. The method of claim 64, wherein the inputvariable is an organic compound.
 67. The method of claim 64, wherein theinput variable is an inorganic compound.
 68. The method of claim 64,wherein the input variable is a complex sample.
 69. The method of claim64, wherein the input variable is a pharmaceutical, environmentalsample, a nutritional sample, or a consumer product.
 70. The method ofclaim 64, wherein the input variable is a virus, liposome, nanoparticle,biodegradable polymer, radiolabeled particle or toxin, biomolecul,toxin-conjugated particle or biomolecule.
 71. The method of claim 64,wherein the input variable is a stabilizing agent.
 72. The method ofclaim 71, wherein the stabilizing agent is albumin, polyethyleneglycol,poly(ethylene-co-vinyl acetate), or poly(lactide-co-glycolide).
 73. Apharmacokinetic-based microscale culture device comprising: a firstmicroscale chamber containing a cell culture comprising a first type ofcell under physiological conditions where the cell culture provides atleast one pharmacokinetic parameter value comparable to a value obtainedfor the same type of cell culture in vivo, wherein the first chambercomprises a first inlet and a first outlet for flow of culture medium;and a sensor for obtaining signals from the cell culture.
 74. Theculture device of claim 73, wherein the cell culture further comprisesat least one additional type of cell.
 75. The culture device of claim73, further comprising: a second microscale chamber containing a secondtype of cell culture under conditions where the second type of cellculture provides at least one pharmacokinetic parameter value comparableto a value obtained for the same type of cell in vivo, wherein thesecond chamber comprises a second inlet and a second outlet for flow ofculture medium; a sensor for obtaining signals from the cell culture;and a microfluidic channel interconnecting the first and secondchambers.
 76. The culture device of claim 73, further comprising culturemedium.
 77. The culture device of claim 76, wherein the culture mediumflows Through the chambers.
 78. The culture device of claim 76, whereinthe culture medium flows re-circulated through the chamber.
 79. Theculture device of claim 73 further comprising a pumping mechanism. 80.The culture device of claim 79, wherein the pumping mechanism isintegrated in the device.
 81. The culture device of claim 79, whereinthe pumping mechanism is external to the device.
 82. The culture deviceof claim 73, further comprising a debubbler located within themicrofluidic channel.
 83. The culture device of claim 73, furthercomprising a debubbler located externally to the device.
 84. The culturedevice of claim 73, wherein at least one of the pharmacokineticparameters is a measurement of interaction between cells, liquidresidence time, liquid to cell ratio, metabolism by cells, or shearstress.
 85. The culture device of claim 73, wherein the sensor is abiosensor.
 86. The culture device of claim 73, wherein the sensorcomprises a waveguide.
 87. The culture device of claim 73, wherein thedevice is microfabricated.
 88. The culture device of claim 73, whereinthe device is manufactured from a microfabricated master.
 89. Theculture device of claim 73, wherein the chamber provides forthree-dimensional growth of cells.
 90. The culture device of claim 73,wherein the chamber contains a plurality of cells.
 91. The culturedevice of claim 73, wherein the chamber contains a tissue biopsy. 92.The culture device of claim 73, wherein the chamber contains across-section of a tissue.
 93. The culture device of claim 91 or 92,wherein the tissue is healthy or diseased.
 94. The culture device ofclaim 91 or 92, wherein the tissue is an artery, vein, gastrointestinaltract, esophagus, or colon.
 95. The culture device of claim 73, whereinthe chamber contains a cross-section of an organ.
 96. The culture deviceof claim 95, wherein the organ is healthy or diseased.
 97. The culturedevice of claim 95, wherein the organ is a heart, brain, kidney, lung,or muscle.
 98. The culture device of claim 73, wherein the chambercontains circulating or adherent cells.
 99. The culture device of claim73, wherein the chamber contains eukaryotic cells.
 100. The culturedevice of claim 99, wherein the eukaryotic cells are plant or animalcells.
 101. The culture device of claim 100, wherein the cells aremammalian cells.
 102. The culture device of claim 73, wherein thechamber contains prokaryotic cells.
 103. The culture device of claim 73,wherein the cells are primary cells.
 104. The culture device of claim73, wherein the cells are tumor cells.
 105. The culture device of claim73, wherein the cells are stem cells.
 106. The culture device of claim73, wherein the cells are genetically altered, transformed, orimmortalized cells.
 107. The method for producing a biological productcomprising providing the culture device of claim 1 or 73 containing aculture medium, and a cell that generates the biological product. 108.The method of claim 107, wherein the cell is a host cell geneticallyengineered to produce an exogenous gene product.
 109. The method ofclaim 107, wherein the biological product is a growth factor, regulatoryfactor, peptide hormone, or antibody.
 110. The method of claim 107,further comprising isolating the biological product from the culturemedium using a standard isolation technique.
 111. The method of claim110, wherein the isolation technique is HPLC, column chromatography, orelectrophoresis.
 112. A microscale culture device, comprising: a firstmicroscale chamber having a geometry simulating a first in vivointeraction with culture medium, wherein the first chamber comprises afirst inlet and a first outlet for flow of the culture medium; a secondmicroscale chamber having a geometry simulating a second in vivointeraction with the culture medium, wherein the second chambercomprises a second inlet and a second outlet for flow of the culturemedium; and a microfluidic channel interconnecting the first and secondchambers.
 113. The microscale culture device of claim 112 wherein thechambers are formed from a plastic material.
 114. The microscale culturedevice of claim 113 wherein the plastic material is selected from thegroup consisting of polystryrene, polymethylmethacrylate, polycarbonate,polytetrafluoroethylene, polyvinylchloride, polydimethylsiloxane, andpolysulfone.
 115. The microscale culture device of claim 112 and furthercomprising a pump coupled to the first inlet.
 116. The array ofmicroscale culture devices of claim 115 wherein the pump is selectedfrom the group consisting of a peristaltic pump, diaphragm pump, ormicroelectromechanical pump.
 117. The microscale culture device of claim115 wherein the pump is also coupled to the second outlet to recirculateculture medium.
 118. The microscale culture device of claim 112 whereinthe chamber geometries are based on a mathematical model representingorgans of a living body.
 119. The microscale culture device of claim 118wherein the model is a physiological-based pharmacokinteic model. 120.The microscale culture device of claim 119 wherein the model simulatesat least one of known tissue size ratio, tissue to blood volume ratio,and drug residence time.
 121. The microscale culture device of claim 112and further comprising sensors for measuring physiological events in thechambers.
 122. The microscale culture device of claim 121 wherein thephysiological events comprise cell death, cell proliferation,differentiation, immune response, or perturbations in metabolism orsignal transduction pathways.
 123. The microscale culture device ofclaim 121 wherein pharmacokinetic data is derived from the sensors. 124.The microscale culture device of claim 121 wherein the sensors areintegrated with the device and provide real-time readout of thephysiological status of the cells in the system.
 125. A microscaleculture device comprising: a plurality of chambers connected by fluidicpassages, each chamber having a geometry simulating parts of a livingbody; and a pump, for circulating culture medium through the chambers tosimulate the effects of compounds on the living body.
 126. Themicroscale culture device of claim 125 wherein the pump is a peristalticpump that recirculates the culture medium through the chambers.
 127. Themicroscale culture device of claim 125 wherein the chambers simulateinteraction of the culture medium with at least two of a liver, lung, anarea of slowly perfused fluid, fat, and an area of rapidly perfusedfluid.
 128. The microscale culture device of claim 125 wherein onechamber simulates a lung with multiple parallel ridges of material. 129.The microscale culture device of claim 125 wherein one chamber simulatesa liver with multiple staggered pillars.
 130. The microscale culturedevice of claim 125 and further comprising a controller.
 131. Themicroscale culture device of claim 130 wherein the controller controlsthe pump to create culture medium residences times in chamberscomparable to those encountered in the living body.
 132. The microscaleculture device of claim 131 and further comprising valves distributedalong the fluid passages, and wherein the controller controls the valvesconsistent with pharmacokinetic parameter values associated with thesimulated parts of the living body.
 133. The microscale culture deviceof claim 130 wherein the chambers are formed on a substrate, and thecontroller is separate from and electrically coupled to the substrate.134. The microscale culture device of claim 130 and further comprising alook-up table having pharmacokinetic parameter values associated withthe simulated parts of the living body for use by the controller.
 135. Amicroscale culture device, comprising: a lung simulating chamber; apump; at least two of a liver simulating chamber, a slowly perfusedsimulating chamber, a rapidly perfused simulating chamber and a fatsimulating chamber coupled in parallel; and a plurality of microfluidicchannels serially coupling the lung simulating chamber, the pump, andthe at least two chambers.
 136. A method of forming a microscale culturedevice, the method comprising: forming a first microscale chamber havinga geometry simulating a first in vivo interaction with culture medium,wherein the first chamber comprises a first inlet and a first outlet forflow of the culture medium; forming a second microscale chamber having ageometry simulating a second in vitro interaction with the culturemedium, wherein the second chamber comprises a second inlet and a secondoutlet for flow of the culture medium; and forming a microfluidicchannel interconnecting the first and second chambers.
 137. The methodof claim 136 wherein the chambers are formed by embossing, injectionmolding, or stamping.
 138. The method of claim 136 and furthercomprising polymerizing surfaces of the chambers and channel.
 139. Themethod of claim 138 wherein polymeric materials on the surface of thechambers and channels provide enhanced fluid direction, cellularattachment or cellular segregation.
 140. An array of microscale culturedevices comprising: a housing for enclosing the devices, each devicecomprising: a first microscale chamber having a geometry simulating afirst in vivo interaction with culture medium, wherein the first chambercomprises a first inlet and a first outlet for flow of the culturemedium; and channels coupled to the inlets and outlets of the chamber.141. The array of microscale culture devices of claim 140 wherein eachdevice further comprises a second microscale chamber having a geometrysimulating a second in vitro interaction with the culture medium,wherein the second chamber comprises a second inlet and a second outletfor flow of the culture medium with channels coupled thereto.
 142. Thearray of microscale culture devices of claim 140 wherein the devices arecoupled in parallel or multiplexed to simulate biological barriers. 143.The array of microscale culture devices of claim 142 wherein thebarriers are gastrointestinal barriers or the blood brain barrier. 144.The array of microscale culture devices of claim 140, wherein the numberof devices is greater than approximately
 10. 145. A system comprising: afirst microscale culture device having a plurality of microscalechambers with geometries that simulate a plurality of in vivointeractions with a culture medium, wherein each chamber includes aninlet and an outlet for flow of the culture medium, and a microfluidicchannel interconnecting the chambers; and a control instrument coupledto the first microscale culture device, the control instrument having acomputer to acquire data from, and control pharmacokinetic parametersof, the first microscale culture device.
 146. The system of claim 145,wherein the first microscale culture device is formed on a computerizedchip.
 147. The system of claim 145, wherein the first microscale culturedevice further includes one or more sensors coupled to the controlinstrument for measuring physiological events in the chambers.
 148. Thesystem of claim 147, wherein the sensors include one or more biosensorsthat monitor the oxygen, carbon dioxide, or pH of the culture medium.149. The system of claim 145, wherein the control instrument holds thefirst microscale culture device, and seals a top of the first microscaleculture device to establish the microfluidic channel.
 150. The system ofclaim 145, wherein the control instrument further includes a pump tocontrol circulation of microfluid in the microfluidic channel of thefirst microscale culture device, a heating element to control thetemperature of the first microscale culture device, a light source, anda photodetector to detect fluorescent emissions from cell compartmentswithin the first microscale culture device.
 151. The system of claim150, wherein the computer further records data for fluorescentintensity.
 152. The system of claim 151, wherein the computer recordsdata for fluorescent intensity using a measuing instrument of a typethat is selected from a group consisting of colorimetric, fluorometric,luminescent, and radiometric.
 153. The system of claim 150, wherein theheating element maintains the first microscale culture device at atemperature of 37 degrees Celsius.
 154. The system of claim 145, whereinthe computer controls a pharmacokinetic parameter selected from a groupconsisting of group pump speed, temperature, length of experiment, andfrequency of data acquisition of the first microscale culture device.155. The system of claim 145, wherein the computer controls apharmacokinetic parameter selected from a group consisting of flow rate,chamber geometry, and number of cells in the first microscale culturedevice.
 156. The system of claim 145, wherein the computer furthercontrols one or more pumps in the first microscale culture device tocreate culture medium residence times in the chambers comparable tothose encountered in the living body.
 157. The system of claim 145,wherein the computer further controls one or more valves distributedalong the microfluidic channel in a manner that is consistent with apharmacokinetic parameter value associated with a simulated part of aliving body.
 158. The system of claim 145, and further comprising asecond microscale culture device having a plurality of microscalechambers with geometries that simulate a plurality of in vivointeractions with a culture medium, wherein each chamber includes aninlet and an outlet for flow of the culture medium, and a microfluidicchannel interconnecting the chambers, wherein the control instrument iscoupled to the second microscale culture device.
 159. A computerizedmethod for dynamically controlling a microscale culture device, thecomputerized method comprising: analyzing data from a plurality ofsensors to measure physiological events in a plurality of chambers ofthe microscale culture device; regulating fluid flow rates of a culturemedium in the chambers of the microscale culture device; detectingbiological or toxicological reactions in the chambers of the microscaleculture device; and upon detection, charging one or more pharmacokineticparameters of the microscale culture device.
 160. The computerizedmethod of claim 159, wherein the detecting includes detecting a changein dimension of a cell compartment of the microscale culture device.161. The computerized method of claim 159, wherein the changing includeschanging a pharmacokinetic parameter selected from a group consisting ofinteractions between cells, liquid residence time, liquid to cellratios, metabolism by cells, and shear stress in the microscale culturedevice.
 162. The computerized method of claim 159, wherein the changingincludes changing a pharmacokinetic parameter selected from a groupconsisting of flow rate, chamber geometry, and number of cells in themicroscale culture device.
 163. The computerized method of claim 159,and further comprising optimizing chamber geometry within the microscaleculture device, wherein the optimizing includes selecting a quantity ofchambers, choosing a chamber geometry that provides a proper tissue ororgan size ratio, choosing an optimal fluid flow rate that provides aproper liquid residence time, and calculating a cell shear stress. 164.The computerized method of claim 159, and further comprising regulatinga temperature of the culture medium.
 165. The computerized method ofclaim 159, and further comprising detecting fluorescent emissions from acell compartment of the microscale culture device.
 166. Acomputer-readable medium having computer-executable instructions storedthereon to perform a method, the method comprising: analyzing data froma plurality of sensors to measure physiological events in a plurality ofchambers of the microscale culture device; regulating fluid flow ratesof a culture medium in the chambers of the microscale culture device;detecting biological or toxicological reactions in the chambers of themicroscale culture device; and upon detection, changing one or morepharmacokinetic parameters of the microscale culture device.
 167. Thecomputer-readable medium of claim 166, wherein the changing includeschanging a pharmacokinetic parameter selected from a group consisting ofinteractions between cells, liquid residence time, liquid to cellratios, metabolism by cells, and shear stress in the microscale culturedevice.
 168. The computer-readable medium of claim 166, wherein thechanging includes changing a pharmacokinetic parameter selected from agroup consisting of flow rate, chamber geometry, and number of cells inthe microscale culture device.
 169. The computer-readable medium ofclaim 166, wherein the method further comprises optimizing chambergeometry within the microscale culture device, and wherein theoptimizing includes selecting a quantity of chambers, choosing a chambergeometry that provides a proper tissue or organ size ratio, choosing anoptimal fluid flow rate that provides a proper liquid residence time,and calculating a cell shear stress.
 170. The computer-readable mediumof claim 166, wherein the method further comprises regulating atemperature of the culture medium.
 171. The computer-readable medium ofclaim 166, wherein the method further comprises detecting fluorescentemissions from a cell compartment of the microscale culture device. 172.A computer comprising: a microprocessor, a general memory; anon-volatile-storage element; an input/output interface that includes aninterface to a microscale culture device having one or more sensors; andcomputer software executable on the microprocessor to analyze data fromthe sensors to measure physiological events in a plurality of chambersof the microscale culture device, regulate fluid flow rates of a culturemedium in the chambers of the microscale culture device, detectbiological or toxicological reactions in the chambers of the microscaleculture device, and upon detection, change one or more pharmacokineticparameters of the microscale culture device.
 173. The computer of claim172, wherein the non-volatile storage element includes historical datataken from published information, data gathered from previously runtests, or data derived from theoretical calculations.
 174. The computerof claim 172, wherein the computer software regulates the fluid flowrates by transmitting commands to one or more pumps of the microscaleculture device through pump control lines.
 175. The computer of claim172, wherein the computer software is further executable on themicroprocessor to regulate a temperature of the culture medium.
 176. Thecomputer of claim 175, wherein the computer software regulates thetemperature by transmitting commands to a heater coil of the microscaleculture device through heater coil control lines.
 177. The computer ofclaim 172, and further comprising; a look-up table memory coupled to thegeneral memory for storing a set of mass balance equations thatrepresent physiologically-based pharmacokinetic models for variousbiological or chemical substances in the system; and a cache memorycoupled to the microprocessor for storing the computer software. 178.The computer of claim 172, wherein the input/output interface furtherincludes a keyboard interface, a display interface, and aprinter/plotter recorder interface.